Methods and compositions for diagnosing and treating prostate cancer based on long noncoding RNA overlapping the LCK gene that regulates prostate cancer cell growth

ABSTRACT

Provided herein is a previously unannotated lncRNA lying within exon six and 3′UTR of the LCK gene, labeled “HULLK” for Hormone-Upregulated lncRNA within LCK. HULLK is a novel lncRNA situated within the LCK gene that can serve as an oncogene in PCa. Accordingly, provided are methods and compositions for diagnosing and treating prostate cancer based on HULLK that regulates prostate cancer cell growth.

CROSS REFERENCE TO RELATED APPLICATION

The presently disclosed subject matter claims the benefit of U.S.Provisional Patent Application Ser. No. 62/889,705, filed Aug. 21, 2019;the disclosure of which is incorporated herein by reference in itsentirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No. CA178338awarded by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD

The presently disclosed subject matter relates to biomarkers andtherapeutic targets of prostate cancer (PCa). In some embodiments, thepresently disclosed subject matter relates to a long noncoding RNA(lncRNA) situated in the LCK gene that can serve as an oncogene in PCa.

Abbreviations

PCa: prostate cancer;

CRPC: castration-resistant prostate cancer;

lncRNA: long noncoding RNA;

qPCR: quantitative PCR;

ddPCR: droplet digital PCR;

LCK: lymphocyte-specific protein tyrosine kinase;

HULLK: hormone-upregulated lncRNA within LCK;

TSS: transcriptional start site;

ncRNA: noncoding RNA;

lncRNA-ATB: long noncoding RNA Activated by Transforming GrowthFactor-Beta; PRUNE2: prune homolog 2;

DANCR: differentiation antagonizing non-protein coding RNA;

PCAT: prostate cancer-associated transcript;

PCA3: Prostate Cancer Antigen 3;

CTBP1-AS: c-terminal binding protein 1-antisense;

HOTAIR: HOX transcript antisense RNA;

NEAT1: nuclear-enriched abundant transcript 1;

SChLAP1: second chromosome locus associated with prostate-1;

GAS5: growth arrest-specific 5;

TMPRSS2: transmembrane protease, serine 2;

MDM2: mouse double minute 2 homolog;

FANTOM: functional annotation of the mammalian genome;

ENCODE: encyclopedia of DNA elements;

EMT: epithelial-to-mesenchymal transition;

BET: bromodomain and extra-terminal;

RACE: rapid amplification of cDNA ends;

FFPE: formalin-fixed paraffin-embedded;

NP: normal prostate;

BCa: breast cancer;

ADT: androgen deprivation therapy;

shRNA: short-hairpin RNA;

EIF3I: Eukaryotic translation initiation factor 3 subunit I;

HDAC1: histone deacetylase 1

BACKGROUND

One in forty-one men diagnosed with metastatic prostate cancer (PCa)will die from the disease. While androgen deprivation therapy (ADT) isthe current initial treatment for advanced PCa, eventually all mendiagnosed with PCa will develop incurable castration-resistant prostatecancer (CRPC).

Next-generation sequencing of the PCa transcriptome has uncovered thatapproximately a quarter of abundant transcripts are long noncoding RNAs(lncRNAs), suggesting that they may play a larger role in PCa thanoriginally thought (1). In fact, PCa-specific lncRNAs have been reportedfor every stage in the progression of the disease. Therefore, lncRNAsmay serve as therapeutic targets for combating PCa progression.Unfortunately, despite the growing list of annotated lncRNAs predictedby RNA sequencing, the number of lncRNAs that have been thoroughlydefined and experimentally verified is very small. It is presumable thatthousands of lncRNAs remain to be detected since those arising fromoverlapping protein-coding loci still need to be analyzed (16).

Therefore, there exists a serious need for more effective therapies forthe treatment of advanced PCa, and that requires a more completeunderstanding of PCa development and progression.

SUMMARY

This summary lists several embodiments of the presently disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently disclosed subjectmatter, whether listed in this summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

In some embodiments, provided herein are methods of modulatinglymphocyte-specific protein tyrosine kinase (LCK) activity in avertebrate subject. The methods can in some embodiments compriseadministering to the vertebrate subject an effective amount of asubstance capable of modulating expression of an LCK gene in thevertebrate subject, wherein the substance comprises an RNA interference(RNAi) molecule directed to the LCK gene, whereby modulation of LCKactivity is accomplished. In some embodiments, modulating expression ofthe LCK gene comprises modulating expression of a long noncoding RNA(lncRNA) of the LCK gene. The lncRNA can comprise a hormone upregulatedlncRNA within the LCK gene (HULLK).

In some embodiments, HULLK can comprise a nucleotide sequence having atleast about 75% sequence identity to SEQ ID NO. 1. In some aspects, theRNAi molecule comprises a short hairpin RNA (shRNA), whereby the shRNAmodulates expression of the LCK gene by RNAi. In some aspects, the shRNAcan comprises shLCK-3 and/or shLCK-4. The shRNA can be configured totarget a carboxy-terminal of the LCK gene.

In some aspects, the substance further comprises an anti-androgencompound, optionally wherein the anti-androgen compound is selected fromthe group consisting of enzalutamide, an inhibitor of p300, an inhibitorof the bromodomain family, and an inhibitor of the extra-terminal (BET)family. The substance can further comprise a delivery vehicle, such asbut not limited to a viral vector, an antibody, an aptamer or ananoparticle, for delivering the shRNA to a target cell.

In some aspects, the RNAi molecule can comprise a small interfering RNA(siRNA), whereby the siRNA modulates expression the LCK gene by RNAi.The substance can further comprises a delivery vehicle, wherein thedelivery vehicle is selected from a viral vector, an antibody, anaptamer, or a nanoparticle for delivering the siRNA to a target cell.The substance can be configured to target HULLK in cytoplasm of a cellin the vertebrate subject.

In some aspects, modulating expression of the LCK gene can modulategrowth and/or proliferation of a prostate cancer (PCa) cell within thevertebrate subject. The vertebrate subject can in some aspects besuffering from PCa, wherein the PCa comprises androgen-dependent PCaand/or castration-resistant PCa.

Also provided herein in some embodiments are compositions for modulatingexpression of hormone upregulated long noncoding RNA within LCK gene(HULLK), the compositions comprising an RNAi construct configured tomodulate expression of HULLK. The RNAi can comprise a siRNA, shRNA,miRNA, or ribozyme. The siRNA, shRNA, miRNA, or ribozyme can be specificfor a vertebrate HULLK comprising a nucleotide sequence of SEQ ID NO. 1or variant thereof, wherein the variant has at least about 75% sequenceidentity to SEQ ID NO. 1. In some aspects, the compositions can furthercomprise a pharmaceutically acceptable carrier.

Provided are expression vectors comprising a nucleic acid sequenceencoding an RNAi construct as disclosed herein. The expression vectorcan in some embodiments be a retroviral vector. Mammalian cellscomprising such an expression vector are also provided.

In some embodiments, provided herein are methods of diagnosing a cancerin a subject. Such methods can comprise providing a sample from asubject, analyzing the sample with or without prior concentration of thesample to determine the presence of and/or expression level of hormoneupregulated long noncoding RNA within LCK gene (HULLK) in the sample,and diagnosing the subject as having a cancer based on the detection ofand/or expression level of HULLK in the sample. Diagnosing a cancer cancomprise diagnosing a prostate cancer (PCa).

Such methods can in some aspects further comprise determining thepresence of cytoplasmic HULLK in a sample from the subject, whereinidentifying the presence of cytoplasmic HULLK is indicative ofmetastatic PCa. In some embodiments, the subject is a human subjectsuspected of having PCa.

These and other objects are achieved in whole or in part by thepresently disclosed subject matter. Further, other objects andadvantages of the presently disclosed subject matter will becomeapparent to those skilled in the art after a study of the followingdescription, Drawings and Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The presently disclosed subject matter can be better understood byreferring to the following figures. The components in the figures arenot necessarily to scale, emphasis instead being placed uponillustrating the principles of the presently disclosed subject matter(often schematically). In the figures, like reference numerals designatecorresponding parts throughout the different views. A furtherunderstanding of the presently disclosed subject matter can be obtainedby reference to an embodiment set forth in the illustrations of theaccompanying drawings. Although the illustrated embodiment is merelyexemplary of systems for carrying out the presently disclosed subjectmatter, both the organization and method of operation of the presentlydisclosed subject matter, in general, together with further objectivesand advantages thereof, may be more easily understood by reference tothe drawings and the following description. The drawings are notintended to limit the scope of this presently disclosed subject matter,which is set forth with particularity in the claims as appended or assubsequently amended, but merely to clarify and exemplify the presentlydisclosed subject matter. For a more complete understanding of thepresently disclosed subject matter, reference is now made to thefollowing drawings in which:

FIGS. 1A to 1D illustrate the discovery of a novel lncRNA in prostatecancer cells. FIG. 1A shows the results of LCK protein expression in PCacells in response to serum starvation or hormone supplementation. LCKprotein was immunoprecipitated from 1 mg cell lysate from LNCaP, C4-2,and Jurkat cells cultured in the appropriate growth media for 48 hrs andtreated with 1 nM R1881 or starved of hormone for 24 hrs, separated by10% SDS-PAGE, and immunoblotted with LCK antibodies targeting the amino-and carboxy-terminals. Representative blots are shown, n=3. FIG. 1Bshows results of the inhibition of the proteasome does not reveal LCKprotein expression. LNCaP cells were seeded and treated with 10 μM MG132in the presence or absence of R1881 for the indicated timepoints. Celllysates were blotted for LCK (c-terminal), LCK (n-terminal), p53,ERK1/2, Ran, and Histone H3. Representative blots are shown, n=3. 5′/3′RACE was performed to determine the sequence of a candidate RNA speciesas shown in FIG. 1C. Representative gel is shown, n=3. LNCaP cells weregrown in the absence or presence of 1 nM R1881 for 24 hrs. RNA wascollected using the Qiagen RNeasy kit. 5′/3′ RACE identified a candidatelncRNA spanning exon 6 through the 3′UTR of LCK as shown in FIG. 1D.

FIGS. 2A through 2E illustrate that HULLK is an AR-regulated lncRNA.FIG. 1A shows that HULLK is positively regulated by androgen, n=3. LNCaPand C42 cells were transduced with two independent shRNAs to LCK andtreated with 0-1 nM R1881. RNA was collected on Day 4 and LCK transcriptlevels were determined using LCK primers targeting the 3′UTR. FIG. 2Bshows that Enzalutamide blocks the androgen-induced expression of HULLK,n=3. LNCaP and C42 cells were seeded for 48 hrs, and then, treated with1 nM R1881 in the presence or absence of 10 μM enzalutamide. RNA wascollected 24 hrs after treatment and LCK transcript levels weredetermined by qPCR using LCK primers targeting the 3′UTR. HULLK is notupregulated in AR-null PCa cells, n=3, as shown in FIG. 2C. C4-2, DU145,and PC3 cells were seeded in CSS media for 48 hrs, and then, treatedwith 1 nM R1881 for 16 hrs. RNA was collected and LCK transcript levelswere determined using LCK primers targeting the 3′UTR. In FIG. 2D thedata show that inhibition of p300 blocks the increase in HULLKexpression induced by hormone, n=3. FIG. 2E illustrates that Brd4inhibition reduces the hormone induction of HULLK, n=3. LNCaP, C42, orJurkat cells were seeded for 48 hrs in the appropriate growth media, andthen, treated with 1 nM R1881 in the presence or absence of 0.3 μM A-485or 0.1 μM JQ1. RNA was collected 24 hrs after treatment and LCKtranscript levels were determined by qPCR using LCK primers targetingexon 13 or 3′UTR. PSA and TMPRSS2 transcripts were determined to verifythe efficacy of p300 and Brd4 inhibition.

FIGS. 3A and 3B illustrate the results of studies designed tocharacterize HULLK. As shown in FIG. 3A, HULLK is transcribed from thesense strand of DNA. LNCaP cells were seeded for 48 hrs and treated with1 nM R1881 for 24 hrs. RNA was collected and cDNA was synthesized usingstrand-specific primers. qPCR was performed with two independent LCKprimer sets that amplify HULLK, n=3. As shown in FIG. 3B, HULLK islocalized to the cytoplasm. LNCaP cells were seeded for 48 hrs andtreated with 1 nM R1881 for 24 hrs. Whole cell lysates were separatedinto cytoplasmic and nuclear fractions by centrifugation. RNA wascollected from each fraction and cDNA was synthesized using the iScriptcDNA synthesis kit. qPCR was performed with two independent LCK primersets that amplify HULLK, and cytoplasmic and nuclear control genes, n=3.

FIGS. 4A through 4G illustrate that HULLK expression is present inprostate cancer. FIG. 4A shows the expression of HULLK in a panel of PCaand normal prostate epithelial cell lines cultured in complete growthmedia. qPCR was performed with two independent LCK primer sets targetingexon 2 (E2) and the 3′UTR (3U), n=3. HULLK expression in C4-2, MCF7,BT549, MDA-MB-231, HeLa, and PANC1 cell lines grown in complete growthmedia is shown in FIG. 4B. qPCR was performed with LCK primers targetingthe 3′UTR, n=3. The data plotted in FIG. 4C shows a significant positivecorrelation between HULLK expression and PCa grade. 33 FFPE tissuesamples were obtained from the Biorepository and Tissue ResearchFacility at the University of Virginia. These samples represent biopsiesand surgical resections collected in 2016 from PCa patients presentingwith Gleason Score 6-10. Normal and cancerous regions were demarcatedand 1.5 mm punches were collected. ddPCR was performed on the QX200droplet digital PCR system (Bio-Rad). The data plotted in FIG. 4D isbased on 16 fresh-frozen tissue samples from Gleason Score 6-10 PCapatients were acquired from Dr. Ganesh Raj from the University of TexasSouthwestern Medical Center. These samples were surgically resected fromMay to December in 2017, placed in RNALater (ThermoFisher), and frozenimmediately. FIG. 4E summarizes the statistical analysis of theassociation between the LCK E13 to LCK E4 ratio and grade was performedusing linear regression models, following transformation to the logscale. The analyses were conducted separately for frozen and FFPEsamples, with a test for interaction used to test whether theassociation between the E13 to E4 ratio and grade was the same in frozensamples as in FFPE samples. (F) HULLK expression in normal and tumor(FIG. 4F) and low Gleason [scores 6 and 7(3+4)] and high Gleason score[scores 7(4+3), and 8-10] (FIG. 4G) in the PRAD TCGA cohort.

FIGS. 5A through 5D demonstrate that HULLK positively regulates prostatecancer cell growth. FIG. 5A illustrates the gene structure of LCK andlocation of each shRNA targeting LCK. The percent knockdown of HULLK orLCK message, n=3, is shown in FIG. 5B. MISSION shRNAs targeting humanLCK (NM_005356) and the pLKO vector control were purchased fromThermoFisher. Each shRNA was validated for efficiency of LCK or HULLKknockdown in Jurkat and PCa cells by qPCR. (C) The results of CyQuantAssay measuring DNA content as a surrogate for cell number 7 days aftershRNA transduction is shown in FIG. 5C. Growth was compared to untreatedempty vector control and the values were averaged across biologicalreplicates. Error bars represent standard error of the mean. Therelative effect of four independent shRNAs on cell growth in LNCaP, C42,and Rv1 cells in the absence or presence of 0.05 nM R1881, n=3. FIG. 5Dplots the data for the relative effect of HULLK overexpression on cellgrowth in LNCaP, C42, and Rv1 cells in the absence or presence of 0.05nM R1881, n=3. Statistical analysis was performed using ANOVA and Tukeytest.

FIGS. 6A and 6B illustrate that HULLK expression does not affect ARexpression. FIG. 6A shows the expression of AR following HULLKknockdown. LNCaP and C4-2 cells were transduced with vector, shLCK(n-term), or shLCK (c-term) in the appropriate growth media, and wholecell lysates were collected 48 hrs later and blotted for AR21 andERK1/2, n=3. FIG. 6B shows the expression of AR following HULLKoverexpression. LNCaP and C4-2 cells were transduced with vector orHULLK in the appropriate growth media, and whole cell lysates werecollected 48 hrs later and blotted for AR21 and α-tubulin, n=3. RNA wascollected and AR transcript levels were determined using AR primerstargeting the DNA binding domain, n=3.

FIGS. 7A and 7B illustrate the regulation of HULLK by the AR in breastcancer cells. FIG. 7A shows the expression of AR in breast cancer celllines. LNCaP, MCF7, BT549, and MDA-MD-231 cells were seeded in theappropriate growth media, and whole cell lysates were collected 48 hrslater and blotted for AR21 and ERK1/2. FIG. 7B shows HULLK expressionincreases in response to hormone. MCF7, BT549, and MDA-MB-231 cells wereseeded in CSS media for 48 hrs, and then, treated with 1 nM R1881 for 16hrs. RNA was collected and LCK transcript levels were determined usingLCK primers targeting the 3′UTR, n=3.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter, in which some, but not all embodiments of the presentlydisclosed subject matter are described. Indeed, the presently disclosedsubject matter can be embodied in many different forms and should not beconstrued as limited to the embodiments set forth herein; rather, theseembodiments are provided so that this disclosure will satisfy applicablelegal requirements.

I. Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the presentlydisclosed subject matter.

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

All technical and scientific terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. References to techniques employedherein are intended to refer to the techniques as commonly understood inthe art, including variations on those techniques or substitutions ofequivalent techniques that would be apparent to one of skill in the art.

Accordingly, for the sake of clarity, this description will refrain fromrepeating every possible combination of the individual steps in anunnecessary fashion. Nevertheless, the specification and claims shouldbe read with the understanding that such combinations are entirelywithin the scope of the invention and the claims.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a cell” includes aplurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about”. Accordingly, unless indicated to the contrary, thenumerical parameters set forth in this specification and attached claimsare approximations that can vary depending upon the desired propertiessought to be obtained by the presently disclosed subject matter.

As used herein, the term “about,” when referring to a value or to anamount of a composition, dose, sequence identity (e.g., when comparingtwo or more nucleotide or amino acid sequences), mass, weight,temperature, time, volume, concentration, percentage, etc., is meant toencompass variations of in some embodiments ±20%, in some embodiments±10%, in some embodiments ±5%, in some embodiments ±1%, in someembodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethods or employ the disclosed compositions.

The term “comprising”, which is synonymous with “including” “containing”or “characterized by” is inclusive or open-ended and does not excludeadditional, unrecited elements or method steps. “Comprising” is a termof art used in claim language which means that the named elements areessential, but other elements can be added and still form a constructwithin the scope of the claim.

As used herein, the phrase “consisting of” excludes any element, step,or ingredient not specified in the claim. When the phrase “consists of”appears in a clause of the body of a claim, rather than immediatelyfollowing the preamble, it limits only the element set forth in thatclause; other elements are not excluded from the claim as a whole.

As used herein, the phrase “consisting essentially of” limits the scopeof a claim to the specified materials or steps, plus those that do notmaterially affect the basic and novel characteristic(s) of the claimedsubject matter.

With respect to the terms “comprising”, “consisting of”, and “consistingessentially of”, where one of these three terms is used herein, thepresently disclosed and claimed subject matter can include the use ofeither of the other two terms.

As used herein, the term “and/or” when used in the context of a listingof entities, refers to the entities being present singly or incombination. Thus, for example, the phrase “A, B, C, and/or D” includesA, B, C, and D individually, but also includes any and all combinationsand subcombinations of A, B, C, and D.

The term “gene” refers broadly to any segment of DNA associated with abiological function. A gene can comprise sequences including but notlimited to a coding sequence, a promoter region, a cis-regulatorysequence, a non-expressed DNA segment that is a specific recognitionsequence for regulatory proteins, a non-expressed DNA segment thatcontributes to gene expression, a DNA segment designed to have desiredparameters, or combinations thereof. A gene can be obtained by a varietyof methods, including cloning from a biological sample, synthesis basedon known or predicted sequence information, and recombinant derivationof an existing sequence.

The terms “modulate” or “alter” are used interchangeably and refer to achange in the expression level of a gene, or a level of RNA molecule orequivalent RNA molecules encoding one or more proteins or proteinsubunits, or activity of one or more proteins or protein subunits is upregulated or down regulated, such that expression, level, or activity isgreater than or less than that observed in the absence of the modulator.For example, the terms “modulate” and/or “alter” can mean “inhibit” or“suppress”, but the use of the words “modulate” and/or “alter” are notlimited to this definition.

As used herein, the terms “inhibit”, “suppress”, “repress”,“downregulate”, “loss of function”, “block of function”, and grammaticalvariants thereof are used interchangeably and refer to an activitywhereby gene expression (e.g., a level of an RNA encoding one or moregene products) is reduced below that observed in the absence of acomposition of the presently disclosed subject matter. In someembodiments, inhibition results in a decrease in the steady state levelof a target RNA.

The term “RNA” refers to a molecule comprising at least oneribonucleotide residue. By “ribonucleotide” is meant a nucleotide with ahydroxyl group at the 2′ position of a D-ribofuranose moiety. The termsencompass double stranded RNA, single stranded RNA, RNAs with bothdouble stranded and single stranded regions, isolated RNA such aspartially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA, as well as altered RNA, or analog RNA, thatdiffers from naturally occurring RNA by the addition, deletion,substitution, and/or alteration of one or more nucleotides. Suchalterations can include addition of non-nucleotide material, for exampleat one or more nucleotides of the RNA. Nucleotides in the RNA moleculesof the presently disclosed subject matter can also comprise non-standardnucleotides, such as non-naturally occurring nucleotides or chemicallysynthesized nucleotides or deoxynucleotides. These altered RNAs can bereferred to as analogs or analogs of a naturally occurring RNA.

The term “promoter” defines a region within a gene that is positioned 5′to a coding region of a same gene and functions to direct transcriptionof the coding region. The promoter region includes a transcriptionalstart site and at least one cis-regulatory element. The term “promoter”also includes functional portions of a promoter region, wherein thefunctional portion is sufficient for gene transcription. To determinenucleotide sequences that are functional, the expression of a reportergene is assayed when variably placed under the direction of a promoterregion fragment.

The terms “active”, “functional” and “physiological”, as used forexample in “enzymatically active”, “functional chromatin” and“physiologically accurate”, and variations thereof, refer to the statesof genes, regulatory components, chromatin, etc. that are reflective ofthe dynamic states of each as they exists naturally, or in vivo, incontrast to static or non-active states of each. Measurements,detections or screenings based on the active, functional and/orphysiologically relevant states of biological indicators can be usefulin elucidating a mechanism, or defining a disease state or phenotype, asit occurs naturally. This is in contrast to measurements taken based onstatic concentrations or quantities of a biological indicator that arenot reflective of level of activity or function thereof.

II. Methods and Compositions for Diagnosing and Treating MetastaticProstate Cancer

Virtually all patients with metastatic prostate cancer (PCa) willrelapse and develop lethal castration-resistant prostate cancer (CRPC).Next-generation sequencing of the PCa transcriptome has uncovered thatapproximately a quarter of abundant transcripts are long noncoding RNAs(lncRNAs), suggesting that they may play a role in PCa. PCa-specificlncRNAs may be involved in some or all stages in the progression of thedisease. Therefore, lncRNAs may serve as therapeutic targets forcombating PCa progression.

In hormone-sensitive PCa, long noncoding RNA Activated by TransformingGrowth Factor-Beta (lncRNA-ATB) upregulates the expression ofepithelial-to-mesenchymal transition (EMT) factors, which is animportant component that promotes CRPC (2). Furthermore, lncRNA-ATBoverexpression increases proliferation through the elevation in cyclinD1 and cyclin E. In addition to playing a role in EMT, Prostate CancerAntigen 3 (PCA3) modulates the expression of several cancer-relatedgenes (vascular endothelial growth factor A, receptor tyrosine-proteinkinase, Bcl-2-associated death promoter, and telomerase reversetranscriptase) and androgen receptor (AR) cofactors (3). PCA3 alsoregulates tumor suppressor prune homolog 2 (PRUNE2) expression byforming a PRUNE2/PCA3 double-stranded RNA complex which results inPRUNE2 translational repression (4). The lncRNA DifferentiationAntagonizing Non-Protein Coding RNA (DANCR) has been shown to counterthe actions of the androgen-AR signaling axis (5), which drivesepithelial cell terminal differentiation in the normal prostate (6) andinhibits invasion and metastasis in PCa (7). Probably functioning as ascaffold, DANCR suppresses differentiation and promotes invasion andmetastasis by negatively regulating tissue inhibitor ofmetalloproteinases 2/3 (TIMP 2/3) expression through enhancer of zestehomolog 2 recruitment (8). The androgen-AR signaling pathway is alsoopposed by Prostate Cancer-Associated Transcript 29 (PCAT29), whichinhibits proliferation and is repressed by the AR (9). Thus, PCaprogression may be driven by these lncRNAs at the hormone-sensitivestage of the disease.

During androgen deprivation therapy (ADT), there are two lncRNAs thathave been reported to be overexpressed and promote progression to CRPC.Prostate Cancer Gene Expression Marker 1, elevated in response to ADT,translocates to the nucleus and binds U2 small nuclear RNA auxiliaryfactor 2 and heterogeneous nuclear ribonucleoprotein A1. This results inthe upregulation of AR splice variant 7 (AR-V7), and ultimately, inducesproliferation (10). C-terminal Binding Protein 1-Antisense (CTBP1-AS)inhibits CTBP1 expression by complexing with the transcriptionalrepressor Polypyrimidine Tract Binding Protein (PTB)-associatingsplicing factor (PSF) and recruiting histone decarboxylase (HDAC)-pairedamphipathic helix protein Sin3a (Sin3A) complexes to the gene promoter(11). Furthermore, CTBP1-AS promotes cell cycle progression andproliferation through the suppression of p53 and mothers againstdecapentaplegic homolog 3 expression. Thus, these two lncRNAs may driveCRPC.

While many lncRNAs have been implicated in PCa progression to CRPC, onlya small subset has been experimentally validated. For example, theproliferative and invasive capacities of CRPC cells are enhanced as aresult of the overexpression of HOX Transcript Antisense RNA (HOTAIR)(12). While the AR usually decreases HOTAIR expression, HOTAIR increasesAR activity by preventing ubiquitination and degradation of the ARthrough the inhibition of mouse double minute 2 homolog (MDM2). Theestrogen receptor α (ERα)-regulated lncRNA Nuclear-Enriched AbundantTranscript 1 (NEAT1) not only controls the levels of specific PCa genes,but it also modulates the expression of the Transmembrane Protease,Serine 2 (TMPRSS2)-ERG fusion gene (13). Moreover, ERG-positive CRPCfrequently overexpresses PCAT5 (14). Depletion of PCAT5 from PC3 cellsdiminishes proliferation and invasion and induces apoptosis. Similar toPCAT5, Second Chromosome Locus Associated with Prostate-1 (SChLAP1) alsocorrelates with ERG-positive PCa (15). SChLAP1, which is overexpressedin approximately 25% of all PCa, promotes invasion and migration byinteracting with the SWItch/Sucrose Non-Fermentable-complex and blockingits gene expression regulatory function. Despite the growing list ofannotated lncRNAs predicted by RNA sequencing, the number of lncRNAsthat have been thoroughly defined and experimentally verified is verysmall. It is presumable that thousands of lncRNAs remain to be detectedsince those arising from overlapping protein-coding loci still need tobe analyzed (16). However, prior to the instant disclosure, no lncRNAsuitable for use in treating PCa has been identified.

Disclosed herein is a novel lncRNA encoded within thelymphocyte-specific protein tyrosine kinase (LCK) gene locus in PCacells. As disclosed herein, this lncRNA is regulated by the AR, asexpression was increased in response to hormone and blocked in thepresence of enzalutamide, as well as inhibitors of p300 and thebromodomain and extra-terminal (BET) family. As a result, this lncRNAhas been labelled “HULLK” for Hormone Upregulated lncRNA within LCK. Thepolynucleotide sequence of HULLK is provided herein as SEQ ID NO. 1. Itis transcribed from the sense strand of DNA and localizes to thecytoplasm. HULLK transcripts are not only expressed in PCa cell lines,but also PCa patient tissue. Furthermore, as disclosed herein, HULLKexpression is significantly upregulated with increasing tumor grade.Notably, as demonstrated herein, HULLK knockdown with shRNAssignificantly decreases cellular proliferation in the presence andabsence of hormone. HULLK overexpression hypersensitizes PCa cells toandrogen. Thus, the data included herein indicates that HULLK is alncRNA situated within the LCK gene that functions as a novel positiveregulator of PCa cell growth.

Provided herein are methods of modulating lymphocyte-specific proteintyrosine kinase (LCK) activity in a subject, including for example avertebrate subject or human subject. Such methods can compriseadministering to the vertebrate subject an effective amount of asubstance capable of modulating expression of an LCK gene in thevertebrate subject, wherein the substance can comprise an RNAinterference (RNAi) molecule directed to the LCK gene, wherebymodulation of LCK activity is accomplished. In some aspects, modulatingexpression of the LCK gene comprises modulating expression of a longnoncoding RNA (lncRNA) of the LCK gene. As disclosed herein, the lncRNAcan comprise a hormone upregulated lncRNA within the LCK gene (HULLK).

HULLK comprises a nucleotide sequence of SEQ ID NO. 1, or variantthereof. In some embodiments, such variants can have at least about 50%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to SEQ ID NO. 1.

In some embodiments, the RNAi molecule can comprise a short hairpin RNA(shRNA), whereby the shRNA modulates expression of the LCK gene by RNAi.The shRNA comprises shLCK-3 and/or shLCK-4, optionally wherein the shRNAcomprises a nucleotide sequence of CCGGGGGATCCTGCTGACGGAAATTCTCGAGAATTTCCGTCAGCAGGATCCCTTTTTG (shLCK-3; SEQ ID NO: 25) and/orCCGGTCACATGGCCTATGCACATATCTCGA GATATGTGCATAGGCCATGTGATTTTTG (shLCK-4;SEQ ID NO: 26), or variant thereof (50% to 99% sequence identity asdefined herein). In some aspects, the shRNA can be configured to targeta carboxy-terminal of the LCK gene.

In some aspects, and as discussed further in the Examples, the substanceadministered to the subject can further comprise an anti-androgencompound, optionally wherein the anti-androgen compound is selected fromthe group consisting of enzalutamide, an inhibitor of p300, an inhibitorof the bromodomain family, and an inhibitor of the extra-terminal (BET)family. The substance can further comprise a delivery vehicle, such asbut not limited to a viral vector, an antibody, an aptamer or ananoparticle, for delivering the shRNA to a target cell. The RNAimolecule can comprise a small interfering RNA (siRNA), whereby the siRNAmodulates expression the LCK gene by RNAi.

In some aspects, the substance administered to the subject can furthercomprise a delivery vehicle, such as but not limited to a viral vector,an antibody, an aptamer, or a nanoparticle for delivering the siRNA to atarget cell. The substance is configured to target HULLK in cytoplasm ofa cell in the vertebrate subject.

As discussed herein, by modulating expression of the LCK gene the growthof prostate cancer (PCa) cells or other cancer cells can be modulated inthe subject. The vertebrate subject can be any mammal, including ahuman. The vertebrate subject can be suffering from a form of cancer orrelated condition or disease, including for example PCa. In some aspectsthe subject can be suffering from androgen-dependent PCa and/orcastration-resistant PCa.

Correspondingly, also provided herein are methods of treating subjectsor patients suffering from a cancer, including a prostate cancer (PCa)or related condition. The methods of treatment can comprise providing asubject suffering or believe to be suffering from such a condition andadministering to the subject an effective amount of a substance ortherapeutic composition capable of modulating expression of an LCK genein the subject, whereby modulation of the expression of the LCK gene isaccomplished. Modulating expression of the LCK gene can modulate growthof PCa cells in the subject.

In such treatment methods the substance or therapeutic composition cancomprise an RNA interference (RNAi) molecule directed to the LCK gene,whereby modulation of LCK activity is accomplished. In some aspects,modulating expression of the LCK gene comprises modulating expression ofa long noncoding RNA (lncRNA) of the LCK gene. As disclosed herein, thelncRNA can comprise a hormone upregulated lncRNA within the LCK gene(HULLK).

In such treatment or therapeutic methods HULLK can comprise a nucleotidesequence of SEQ ID NO. 1, or variant thereof. In some embodiments, suchvariants can have at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toSEQ ID NO. 1.

In some embodiments of treatment methods the RNAi molecule can comprisea short hairpin RNA (shRNA), whereby the shRNA modulates expression ofthe LCK gene by RNAi. The shRNA comprises shLCK-3 and/or shLCK-4,optionally wherein the shRNA comprises a nucleotide sequence of SEQ IDNOs: 25 and 26, respectively, or variant thereof (50% to 99% sequenceidentity as defined herein). In some aspects, the shRNA can beconfigured to target a carboxy-terminal of the LCK gene.

In some aspects of the therapeutic or treatment methods the substanceadministered to the subject can further comprise an anti-androgencompound, optionally wherein the anti-androgen compound is selected fromthe group consisting of enzalutamide, an inhibitor of p300, an inhibitorof the bromodomain family, and an inhibitor of the extra-terminal (BET)family. The substance can further comprise a delivery vehicle, such asbut not limited to a viral vector, an antibody, an aptamer or ananoparticle, for delivering the shRNA to a target cell. The RNAimolecule can comprise a small interfering RNA (siRNA), whereby the siRNAmodulates expression the LCK gene by RNAi.

The therapeutic compositions and substances administered to the subjectcan further comprise a delivery vehicle, such as but not limited to aviral vector, an antibody, an aptamer, or a nanoparticle for deliveringthe siRNA to a target cell. The substance is configured to target HULLKin cytoplasm of a cell in the vertebrate subject.

As discussed herein, by modulating expression of the LCK gene the growthof prostate cancer (PCa) cells or other cancer cells can be modulated inthe subject. The vertebrate subject can be any mammal, including ahuman. The vertebrate subject can be suffering from a form of cancer orrelated condition or disease, including for example PCa. In some aspectsthe subject can be suffering from androgen-dependent PCa and/orcastration-resistant PCa. Such methods of treatment can further comprisethe administration of radiation therapy, chemotherapy, gene therapy,immunotherapy, a dietary treatment, or combinations thereof.

A method for modulating expression of an LCK gene in a cell, includingdelivering to the cell an effective amount of a vector comprising apolynucleotide that encodes a siRNA, shRNA, miRNA, or other moleculedirected to an LCK gene and/or that interferes with the expression of anLCK gene, or a small molecule, peptide, antibody or aptamer capable ofinterfering with the LCK gene. Such a method can further comprisemaintaining the cell under conditions sufficient for expression of saidsiRNA, shRNA, miRNA, or a molecule that interferes with the LCK gene bymodulating its expression or otherwise interfering with the same. Insome aspects, modulating expression of the LCK gene comprises modulatingexpression of a long noncoding RNA (lncRNA) of the LCK gene, wherein thelncRNA can comprise a hormone upregulated lncRNA within the LCK gene(HULLK). As discussed further herein, HULLK can comprise a nucleotidesequence of SEQ ID NO. 1, or variants thereof. Such methods ofmodulating gene expression within the cell can include the use of adelivery vehicle, such as but not limited to a viral vector, an aptamer,an antibody or a nanoparticle, for delivering the siRNA, shRNA, and/ormiRNA to the cell. The cell can be a PCa cell or other cancer cell wheremodulating the LCK gene could have a therapeutic effect. In someaspects, the cancer cell can be a primary cancer cell or a cell linerepresenting a primary cancer cell.

Correspondingly, methods are also provided for suppressing the growth ofa cancer cell. Such methods can include similar steps and components asthat noted hereinabove for modulating expression of an LCK gene in acell. In such methods, modulating expression of an LCK gene the cancercell can result in decreased growth of the cancer cell. In such methods,the cell can be in a subject, including a human subject suffering fromcancer, including PCa.

In some embodiments compositions for modulating the expression ofhormone upregulated long noncoding RNA within LCK gene (HULLK) are alsoprovided. Such compositions can comprise an RNAi construct configured tomodulate expression of HULLK. The RNAi can include siRNA, shRNA, miRNA,ribozymes and the like. In some aspects, the siRNA, shRNA, miRNA, orribozymes can be specific for a vertebrate HULLK comprising a nucleotidesequence of SEQ ID NO. 1 or variant thereof, wherein the variant has atleast about 75% sequence identity to SEQ ID NO. 1. As noted herein, suchvariants can have at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toSEQ ID NO. 1. Such compositions can achieve modulation of expression ofHULLK in a subject, tissue, cell or sample, including a downregulationin the gene expression of HULLK of about 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99%, or more.

Also provided are expression vectors comprising a nucleic acid sequenceencoding an RNAi construct disclosed herein. Such expression vectors canbe inducible and/or responsive to inducers, as discussed further herein.The vectors can be any suitable expression vector, including for examplea retroviral vector. Such expression vectors can be within a mammaliancell. Such cells can be a cancer cell, including for example a PCa cell,or other cancer cell where modulating HULLK could have a therapeuticeffect.

Pharmaceutical compositions comprising a therapeutically effectiveamount of a HULLK modulator and a pharmaceutically acceptable diluent orvehicle are provided.

Such compositions can comprise a pharmaceutically acceptable carrier,adjuvant or the like.

In some embodiments, provided herein are methods of diagnosing a cancerin a subject, the methods comprising providing a sample from a subject,analyzing the sample with or without prior concentration of the sampleto determine the presence of and/or expression level of hormoneupregulated long noncoding RNA within LCK gene (HULLK) in the sample,and diagnosing the subject as having a cancer based on the detection ofand/or expression level of HULLK in the sample. Such diagnostic methodscan be suitable for diagnosing a prostate cancer (PCa) using HULLK as abiomarker. Such methods can further comprise determining the presence ofcytoplasmic HULLK in a sample from the subject, wherein identifying thepresence of cytoplasmic HULLK is indicative of metastatic PCa. Suchdiagnostic methods can be suitable for use with human subjects suspectedof having PCa.

III. HULLK Polynucleotide Sequence SEQ ID NO. 1:     CTGGT TCTTCAAGAA CCTGAGCCGC AAGGACGCGGAGCGGCAGCT CCTGGCGCCC GGGAACACTC ACGGCTCCTTCCTCATCCGG GAGAGCGAGA GCACCGCGGG TGAGCGGGCGGCGGTCTCGA CCGGGCGCGG GGGTGCCCCG GGGTGTGCCCGAGGGGGGGC GCAGGGTGAG CCCGAGGTGG AGACACGGGGATCGTTTTCA CTGTCGGTCC GGGACTTCGA CCAGAACCAGGGAGAGGTGG TGAAACATTA CAAGATCCGT AATCTGGACAACGGTGGCTT CTACATCTCC CCTCGAATCA CTTTTCCCGGCCTGCATGAA CTGGTCCGCC ATTACACCAA TGCTTCAGATCGGGCTGTGC ACACGGTTGA GCCGCCCCTG CAGACCCAGAAGCCCCAGAA GCCGTGGTGG GAGGACGAGT GGGAGGTTCCCAGGGAGACG CTGAAGCTGG TGGAGCGGCT GGGGGCTGGAACAGTTCGGGG GGTGTGGAT GGGGTACTAC AACGGGCACACGAAGGTGGC GGTGAAGAGC CTGAAGCAGG GCAGCATGTCCCCGGACGCC TTCCTGGCCG AGGCCAACCT CATGAAGCAGCTGCAACACC AGCGGCTGGT TCGGCTCTAC GCTGTGGTCACCCAGGAGCC CATCTACATC ATCACTGAAT ACATGGAGAATGGGAGTCTA GTGGATTTTC TCAAGACCCC TTCAGGCATCAAGTTGACCA TCAACAAACT CCTGGACATG GCAGCCCAAATTGCAGAAGG CATGGCATTC ATTGAAGAGC GGAATTATATTCATCGTGAC CTTCGGGCTG CCAACATTCT GGTGTCTGACACCCTGAGCT GCAAGATTGC AGACTTTGGC CTAGCACGCCTCATTGAGGA CAACGAGTAC ACAGCCAGGG AGGGGGCCAAGTTTCCCATT AAGTGGACAG CGCCAGAAGC CATTAACTACGGGACATTCA CCATCAAGTC AGATGTGTGG TCTTTTGGGATCCTGCTGAC GGAAATTGTC ACCCACGGCC GCATCCCTTACCCAGGGATG ACCAACCCGG AGGTGATTCA GAACCTGGAGCGAGGCTACC GCATGGTGCG CCCTGACAAC TGTCCAGAGGAGCTGTACCA ACTCATGAGG CTGTGCTGGA AGGAGCGCCCAGAGGACCGG CCCACCTTTG ACTACCTGCG CAGTGTGCTGGAGGACTTCT TCACGGCCAC AGAGGGCCAG TACCAGCCTC AGCCTTGAGA GGCCTTGAGA GGCCCTGGGG TTCTCCCCCTTTCTCTCCAG CCTGACTTGG GGAGATGGAG TTCTTGTGCCATAGTCACAT GGCCTATGCA CATATGGACT CTGCACATGAATCCCACCCA CATGTGACAC ATATGCACCT TGTGTCTGTACACGTGTCCT GTAGTTGCGT GGACTCTGCA CATGTCTTGTACATGTGTAG CCTGTGCATG TATGTCTTGG ACACTGTACAAGGTACCCCT TTCTGGCTCT CCCATTTCCT GAGACCACAGAGAGAGGGGA GAAGCCTGGG ATTGACAGAA GCTTCTGCCCACCTACTTTT CTTTCCTCAG ATCATCCAGA AGTTCCTCAAGGGCCAGGAC TTTATCTAAT ACCTCTGTGT GCTCCTCCTTGGTGCCTGGC CTGGCACACA TCAGGAGTTC AATAAATGTC TGTTGATGAC TGTTGTACA

In SEQ ID NO. 1 above, bold text is exon, underlined text is 3′UTR, andregular text is intronic sequence preserved in two 5′RACE clones.

IV. Nucleic Acids

The term “substantially identical”, as used herein to describe a degreeof similarity between nucleotide sequences, refers to two or moresequences that have in one embodiment at least about least 60%, inanother embodiment at least about 70%, in another embodiment at leastabout 80%, in another embodiment at least about 85%, in anotherembodiment at least about 90%, in another embodiment at least about 91%,in another embodiment at least about 92%, in another embodiment at leastabout 93%, in another embodiment at least about 94%, in anotherembodiment at least about 95%, in another embodiment at least about 96%,in another embodiment at least about 97%, in another embodiment at leastabout 98%, in another embodiment at least about 99%, in anotherembodiment about 90% to about 99%, and in another embodiment about 95%to about 99% nucleotide identity, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms (described herein below under the heading“Comparison of Nucleotide and Amino Acid Sequences”) or by visualinspection. In one embodiment, the substantial identity exists innucleotide sequences of at least about 100 residues, in anotherembodiment in nucleotide sequences of at least about 150 residues, andin still another embodiment in nucleotide sequences comprising a fulllength sequence. The term “full length”, as used herein to refer to acomplete open reading frame encoding, for example, a gene of interestpolypeptide. The term “full length” also encompasses a non-expressedsequence, for example a promoter or an inverted terminal repeatsequence.

In one aspect, polymorphic sequences can be substantially identicalsequences. The term “polymorphic” refers to the occurrence of two ormore genetically determined alternative sequences or alleles in apopulation. An allelic difference can be as small as one base pair.

In another aspect, substantially identical sequences can comprisemutagenized sequences, including sequences comprising silent mutations.A mutation can comprise a single base change.

Another indication that two nucleotide sequences are substantiallyidentical is that the two molecules specifically or substantiallyhybridize to each other under stringent conditions. In the context ofnucleic acid hybridization, two nucleic acid sequences being comparedcan be designated a “probe” and a “target”. A “probe” is a referencenucleic acid molecule, and a “′target” is a test nucleic acid molecule,often found within a heterogeneous population of nucleic acid molecules.A “target sequence” is synonymous with a “test sequence”.

The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex nucleic acid mixture (e.g., total cellular DNA or RNA).

The phrase “hybridizing substantially to” refers to complementaryhybridization between a probe nucleic acid molecule and a target nucleicacid molecule and embraces minor mismatches that can be accommodated byreducing the stringency of the hybridization media to achieve thedesired hybridization.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern blot analysis are both sequence- andenvironment-dependent. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, 1993. Generally, highly stringent hybridization andwash conditions are selected to be about 5° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength and pH. Typically, under “stringent conditions” a probe willhybridize specifically to its target subsequence, but to no othersequences.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor Southern or Northern Blot analysis of complementary nucleic acidshaving more than about 100 complementary residues is overnighthybridization in 50% formamide with 1 mg of heparin at 42° C. An exampleof highly stringent wash conditions is 15 minutes in 0.1×SSC at 65° C.An example of stringent wash conditions is 15 minutes in 0.2×SSC bufferat 65° C. See Sambrook & Russell, 2001, for a description of SSC buffer.Often, a high stringency wash is preceded by a low stringency wash toremove background probe signal. An example of medium stringency washconditions for a duplex of more than about 100 nucleotides is 15 minutesin 1×SSC at 45° C. An example of low stringency wash for a duplex ofmore than about 100 nucleotides is 15 minutes in 4× to 6×SSC at 40° C.For short probes (e.g., about 10 to 50 nucleotides), stringentconditions typically involve salt concentrations of less than about 1 MNa⁺ ion, typically about 0.01 to 1M Na⁺ ion concentration (or othersalts) at pH 7.0-8.3, and the temperature is typically at least about30° C. Stringent conditions can also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2-fold (or higher) than that observed for an unrelated probe inthe particular hybridization assay indicates detection of a specifichybridization.

The following are examples of hybridization and wash conditions that canbe used to identify nucleotide sequences that are substantiallyidentical to reference nucleotide sequences of the presently disclosedsubject matter: in one embodiment a probe nucleotide sequence hybridizesto a target nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO₄, 1 mM EDTA at 50° C. followed by washing in 2×SSC, 0.1% SDS at50° C.; in another embodiment, a probe and target sequence hybridize in7% SDS, 0.5 M NaPO₄, 1 mM EDTA at 50° C. followed by washing in 1×SSC,0.1% SDS at 50° C.; in another embodiment, a probe and target sequencehybridize in 7% SDS, 0.5 M NaPO₄, 1 mM EDTA at 50° C. followed bywashing in 0.5×SSC, 0.1% SDS at 50° C.; in another embodiment, a probeand target sequence hybridize in 7% SDS, 0.5 M NaPO₄, 1 mM EDTA at 50°C. followed by washing in 0.1×SSC, 0.1% SDS at 50° C.; in anotherembodiment, a probe and target sequence hybridize in 7% SDS, 0.5 MNaPO₄, 1 mM EDTA at 50° C. followed by washing in 0.1×SSC, 0.1% SDS at65° C.

A further indication that two nucleic acid sequences are substantiallyidentical is that proteins encoded by the nucleic acids aresubstantially identical, share an overall three-dimensional structure,or are biologically functional equivalents. These terms are definedfurther herein below. Nucleic acid molecules that do not hybridize toeach other under stringent conditions are still substantially identicalif the corresponding proteins are substantially identical. This canoccur, for example, when two nucleotide sequences are significantlydegenerate as permitted by the genetic code.

The term “conservatively substituted variants” refers to nucleic acidsequences having degenerate codon substitutions wherein the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues. See Ohtsuka et al., 1985;Batzer et al., 1991; Rossolini et al., 1994.

The term “subsequence” refers to a sequence of nucleic acids thatcomprises a part of a longer nucleic acid sequence. An exemplarysubsequence is a probe, described herein, or a primer. The term “primer”as used herein refers to a contiguous sequence comprising in oneembodiment about 8 or more deoxyribonucleotides or ribonucleotides, inanother embodiment 10-20 nucleotides, and in yet another embodiment20-30 nucleotides of a selected nucleic acid molecule. The primers ofthe presently disclosed subject matter encompass oligonucleotides ofsufficient length and appropriate sequence so as to provide initiationof polymerization on a nucleic acid molecule of the presently disclosedsubject matter.

The term “elongated sequence” refers to an addition of nucleotides (orother analogous molecules) incorporated into the nucleic acid. Forexample, a polymerase (e.g., a DNA polymerase) can add sequences at the3′ terminus of the nucleic acid molecule. In addition, the nucleotidesequence can be combined with other DNA sequences, such as promoters,promoter regions, enhancers, polyadenylation signals, intronicsequences, additional restriction enzyme sites, multiple cloning sites,and other coding segments.

The term “complementary sequences”, as used herein, indicates twonucleotide sequences that comprise antiparallel nucleotide sequencescapable of pairing with one another upon formation of hydrogen bondsbetween base pairs. As used herein, the term “complementary sequences”means nucleotide sequences which are substantially complementary, as canbe assessed by the same nucleotide comparison set forth above, or isdefined as being capable of hybridizing to the nucleic acid segment inquestion under relatively stringent conditions such as those describedherein. A particular example of a complementary nucleic acid segment isan antisense oligonucleotide.

Nucleic acids of the presently disclosed subject matter can be cloned,synthesized, recombinantly altered, mutagenized, or combinationsthereof. Standard recombinant DNA and molecular cloning techniques usedto isolate nucleic acids are known in the art. Site-specific mutagenesisto create base pair changes, deletions, or small insertions are alsoknown in the art. See e.g., Sambrook & Russell (2001) Molecular Cloning:a Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Silhavy et al. (1984) Experiments with GeneFusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Glover& Hames (1995) DNA Cloning: A Practical Approach, 2nd ed. IRL Press atOxford University Press, Oxford/New York; Ausubel (1995) Short Protocolsin Molecular Biology, 3rd ed. Wiley, New York.

TABLE 1 Functionally Equivalent Codons Amino Acids Codons Alanine Ala AGCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic Acid Asp D GAC GAUGlumatic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly GGGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUULysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU MethionineMet M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCUGlutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU SerineSer S ACG AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine ValV GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

V. Comparison of Nucleotide and Amino Acid Sequences

The terms “identical” or percent “identity” in the context of two ormore nucleotide or polypeptide sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same, when compared andaligned for maximum correspondence, as measured using one of thesequence comparison algorithms disclosed herein or by visual inspection.

The term “substantially identical” in regards to a nucleotide orpolypeptide sequence means that a particular sequence varies from thesequence of a naturally occurring sequence by one or more deletions,substitutions, or additions, the net effect of which is to retainbiological activity of a gene, gene product, or sequence of interest.For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer program, subsequence coordinates are designated if necessary,and sequence algorithm program parameters are selected. The sequencecomparison algorithm then calculates the percent sequence identity forthe designated test sequence(s) relative to the reference sequence,based on the selected program parameters.

Optimal alignment of sequences for comparison can be conducted, forexample by the local homology algorithm of Smith & Waterman, 1981, bythe homology alignment algorithm of Needleman & Wunsch, 1970, by thesearch for similarity method of Pearson & Lipman, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, available from Accelrys Inc.,San Diego, Calif., United States of America), or by visual inspection.See generally, Ausubel, 1995.

An exemplary algorithm for determining percent sequence identity andsequence similarity is the BLAST algorithm, which is described inAltschul et al., 1990. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold. These initial neighborhood word hits act as seeds forinitiating searches to find longer HSPs containing them. The word hitsare then extended in both directions along each sequence for as far asthe cumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when the cumulative alignmentscore falls off by the quantity X from its maximum achieved value, thecumulative score goes to zero or below due to the accumulation of one ormore negative-scoring residue alignments, or the end of either sequenceis reached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength W=11, an expectationE=10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. SeeHenikoff & Henikoff, 2000.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. See e.g., Karlin & Altschul, 1993. One measure ofsimilarity provided by the BLAST algorithm is the smallest sumprobability (P(N)), which provides an indication of the probability bywhich a match between two nucleotide or amino acid sequences would occurby chance. For example, a test nucleic acid sequence is consideredsimilar to a reference sequence if the smallest sum probability in acomparison of the test nucleic acid sequence to the reference nucleicacid sequence is in one embodiment less than about 0.1, in anotherembodiment less than about 0.01, and in still another embodiment lessthan about 0.001.

VI. Gene Therapy Delivery Systems

The presently disclosed subject matter also provides gene therapyconstructs or vectors. The particular vector employed in accordance withthe presently disclosed subject matter is not intended to be alimitation of the disclosed and claimed compositions and methods. Thus,any suitable vector, construct or delivery vehicle as would be apparentto those of skill in the art upon a review of the instant disclosure canbe used within the scope of the presently disclosed subject matter.

The vector can be a viral vector or a non-viral vector. Suitable viralvectors include adenoviruses, adeno-associated viruses (AAVs),self-complementary AAV (scAAV; Buie et al., 2010 Invest Ophthalmol VisSci. 51:236-248), retroviruses, pseudotyped retroviruses, herpesviruses, vaccinia viruses, Semiliki forest virus, and baculoviruses.Suitable non-viral vectors comprise plasmids, water-oil emulsions,polethylene imines, dendrimers, micelles, microcapsules, liposomes, andcationic lipids. Polymeric carriers for gene therapy constructs can beused as described in Goldman et al. (1997) Nat Biotechnol 15:462 andU.S. Pat. Nos. 4,551,482 and 5,714,166. Where appropriate, two or moretypes of vectors can be used together. For example, a plasmid vector canbe used in conjunction with liposomes. Provided in some embodiments ofthe presently disclosed subject matter is the use of an adenovirus, asdescribed further herein below.

Suitable methods for introduction of a gene therapy construct into cellsinclude direct injection into a cell or cell mass, particle-mediatedgene transfer, electroporation, DEAE-Dextran transfection,liposome-mediated transfection, viral infection, and combinationsthereof. A delivery method is selected based considerations such as thevector type, the toxicity of the encoded gene, the condition or tissueto be treated and the site of administration and/or treatment.

Viral Gene Therapy Vectors

In some embodiments viral vectors of the presently disclosed subjectmatter can be disabled, e.g. replication-deficient. That is, they lackone or more functional genes required for their replication, whichprevents their uncontrolled replication in vivo and avoids undesirableside effects of viral infection. In some embodiments, all of the viralgenome is removed except for the minimum genomic elements required topackage the viral genome incorporating the therapeutic gene into theviral coat or capsid. For example, in some embodiments it is desirableto delete all the viral genome except the Long Terminal Repeats (LTRs)or Invented Terminal Repeats (ITRs) and a packaging signal. In the casesof adenoviruses, deletions can be made in the E1 region and optionallyin one or more of the E2, E3 and/or E4 regions. In the case ofretroviruses, genes required for replication, such as env and/or gag/polcan be deleted. Deletion of sequences can be achieved by recombinantapproaches, for example, involving digestion with appropriaterestriction enzymes, followed by religation. Replication-competentself-limiting or self-destructing viral vectors can also be used.

Nucleic acid constructs of the presently disclosed subject matter can beincorporated into viral genomes by any suitable approach known in theart. In some embodiments, such incorporation can be performed byligating the construct into an appropriate restriction site in thegenome of the virus. Viral genomes can then be packaged into viral coatsor capsids by any suitable procedure. In particular, any suitablepackaging cell line can be used to generate viral vectors of thepresently disclosed subject matter. These packaging lines complement thereplication-deficient viral genomes of the presently disclosed subjectmatter, as they include, typically incorporated into their genomes, thegenes which have been deleted from the replication-deficient genome.Thus, the use of packaging lines allows viral vectors of the presentlydisclosed subject matter to be generated in culture.

In some embodiments the vector is an adenoviral vector. By way ofexample and not limitation, adenovirus titration and determination ofinfectivity are described in the Examples below.

Plasmid Gene Therapy Vectors

In some embodiments, a therapeutic gene can be encoded by a nakedplasmid. The toxicity of plasmid DNA is generally low and large-scaleproduction is relatively easy. Plasmid transfection efficiency in vivoencompasses a multitude of parameters, such as the amount of plasmid,time between plasmid injection and electroporation, temperature duringelectroporation, and electrode geometry and pulse parameters (fieldstrength, pulse length, pulse sequence, etc.). The methods disclosedherein can be optimized for a particular application by methods known toone of skill in the art, and the presently disclosed subject matterencompasses such variations. See, e.g., Heller et al. (1996) FEBS Lett389:225-228; Vicat et al. (2000) Hum Gene Ther 11:909-916; Miklavcic etal. (1998) Biophys J 74:2152-2158.

Liposomes

The presently disclosed subject matter also provides for the use of genetherapy constructs comprising liposomes. Liposomes can be prepared byany of a variety of techniques that are known in the art. See, e.g.,Betageri et al., 1993 Liposome Drug Delivery Systems, TechnomicPublishing, Lancaster; Gregoriadis, ed., 1993 Liposome Technology, CRCPress, Boca Raton, Fla.; Janoff, ed. 1999 Liposomes: Rational Design, M.Dekker, New York, N.Y.; Lasic & Martin, 1995 Stealth Liposomes, CRCPress, Boca Raton, Fla.; Nabel, 1997 “Vectors for Gene Therapy” inCurrent Protocols in Human Genetics on CD-ROM, John Wiley & Sons, NewYork, N.Y.; and U.S. Pat. Nos. 4,235,871; 4,551,482; 6,197,333; and6,132,766. Temperature-sensitive liposomes can also be used, for exampleTHERMOSOMES™ as disclosed in U.S. Pat. No. 6,200,598. Entrapment of anactive agent within liposomes of the presently disclosed subject mattercan also be carried out using any conventional method in the art. Inpreparing liposome compositions, stabilizers such as antioxidants andother additives can be used.

Other lipid carriers can also be used in accordance with the presentlydisclosed subject matter, such as lipid microparticles, micelles, lipidsuspensions, and lipid emulsions. See, e.g., Labat-Moleur et al., 1996Gene Therapy 3:1010-1017; U.S. Pat. Nos. 5,011,634; 6,056,938;6,217,886; 5,948,767; and 6,210,707.

Inducible Gene Therapy Vectors

In some instances a continuous un-regulated overexpression of transgeneproducts could result in an unwanted physiological or toxic effect.Thus, in an effort to maximize expression levels of a gene productencoded by a gene therapy vector at a desired site and/or at a desiredtime, and concomitantly minimize the constitutive expression and/orsystemic levels of the same encoded gene product, constructs of thepresently disclosed subject matter can comprise an inducible promoter.As disclosed herein, controlled expression of a therapeutic transgenecan be achieved by employing an inducible vector.

In some embodiments, an insult-induced gene therapy construct isprovided that increases the levels of its therapeutic product when theagent triggering the disease is present, and stops its mode of actionwhen it is no longer needed. By way of example and not limitation, aninducible gene therapy construct of the presently disclosed subjectmatter for treating glaucoma can increase expression of its therapeuticpeptide, e.g. MMP1, when the construct is in the presence of a steroid,and stop or substantially decrease expression of its therapeutic geneupon the removal or clearance of the steroid.

At least one advantage of an inducible vector is that it is active onlywhen the insult-triggered agent is present. Therefore, rather than acoding sequence for a polypeptide of interest being constitutivelyexpressed, it will be expressed only when needed, that is, when atriggering agent, e.g. a steroid, is present. Thus, by way of exampleand not limitation, an inducible gene therapy construct of the presentlydisclosed subject matter expressing RNAi directed to HULLK todownregulate expression of the same.

In some embodiments a gene therapy construct of the presently disclosedsubject matter can comprise a steroid response element (SRE). Due to theinducible nature of the SRE, a vector comprising a selected gene and anSRE will express the gene only when exposed to a steroid. In someembodiments, the SRE is a glucocorticoid response element (GRE).

VII. Pharmaceutical Compositions

The presently disclosed subject matter provides pharmaceuticalcompositions comprising a gene therapy construct or other component ofthe presently disclosed subject matter. In some embodiments, apharmaceutical composition can comprise one or more gene therapyconstructs or components produced in accordance with the presentlydisclosed subject matter.

In some embodiments a pharmaceutical composition can also contain apharmaceutically acceptable carrier or adjuvant for administration ofthe gene therapy construct. In some embodiments, the carrier ispharmaceutically acceptable for use in humans. In some embodiments, thecarrier is pharmaceutically acceptable for use in the eye and associatedocular tissues. The carrier or adjuvant desirably should not itselfinduce the production of antibodies harmful to the individual receivingthe composition and should not be toxic. Suitable carriers can be large,slowly metabolized macromolecules such as proteins, polypeptides,liposomes, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, ammo acid copolymers and inactive virusparticles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonate andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions canadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, can be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated for administration to the patient.

The compositions of the presently disclosed subject matter can furthercomprise a carrier to facilitate composition preparation andadministration. Any suitable delivery vehicle or carrier can be used,including but not limited to a microcapsule, for example a microsphereor a nanosphere (Manome et al. (1994) Cancer Res 54:5408-5413; Saltzman& Fung (1997) Adv Drug Deliv Rev 26:209-230), a glycosaminoglycan (U.S.Pat. No. 6,106,866), a fatty acid (U.S. Pat. No. 5,994,392), a fattyemulsion (U.S. Pat. No. 5,651,991), a lipid or lipid derivative (U.S.Pat. No. 5,786,387), collagen (U.S. Pat. No. 5,922,356), apolysaccharide or derivative thereof (U.S. Pat. No. 5,688,931), ananosuspension (U.S. Pat. No. 5,858,410), a polymeric micelle orconjugate (Goldman et al. (1997) Cancer Res 57:1447-1451 and U.S. Pat.Nos. 4,551,482, 5,714,166, 5,510,103, 5,490,840, and 5,855,900), and apolysome (U.S. Pat. No. 5,922,545).

Suitable formulations of pharmaceutical compositions of the presentlydisclosed subject matter include aqueous and non-aqueous sterileinjection solutions which can contain anti-oxidants, buffers,bacteriostats, bactericidal antibiotics and solutes which render theformulation isotonic with the bodily fluids of the intended recipient;and aqueous and non-aqueous sterile suspensions which can includesuspending agents and thickening agents. The formulations can bepresented in unit-dose or multi-dose containers, for example sealedampoules and vials, and can be stored in a frozen or freeze-dried(lyophilized) condition requiring only the addition of sterile liquidcarrier, for example water for injections, immediately prior to use.Some exemplary ingredients are SDS in the range of in some embodiments0.1 to 10 mg/ml, in some embodiments about 2.0 mg/ml; and/or mannitol oranother sugar in the range of in some embodiments 10 to 100 mg/ml, insome embodiments about 30 mg/ml; and/or phosphate-buffered saline (PBS).Any other agents conventional in the art having regard to the type offormulation in question can be used. In some embodiments, the carrier ispharmaceutically acceptable. In some embodiments the carrier ispharmaceutically acceptable for use in humans. In some embodiments thecarrier is pharmaceutically acceptable for use in the eye and oculartissue.

Pharmaceutical compositions of the presently disclosed subject mattercan have a pH between 5.5 and 8.5, preferably between 6 and 8, and morepreferably about 7. The pH can be maintained by the use of a buffer. Thecomposition can be sterile and/or pyrogen free. The composition can beisotonic with respect to humans. Pharmaceutical compositions of thepresently disclosed subject matter can be supplied inhermetically-sealed containers.

VIII. Subjects

The subject treated in the presently disclosed subject matter isdesirably a human subject, although it is to be understood that theprinciples of the disclosed subject matter indicate that thecompositions and methods are effective with respect to invertebrate andto all vertebrate species, including mammals, which are intended to beincluded in the term “subject”. Moreover, a mammal is understood toinclude any mammalian species in which treatment of ocular conditions ortreatment or prevention of glaucoma is desirable, particularlyagricultural, and domestic mammalian species. The methods of thepresently disclosed subject matter are particularly useful in thetreatment of warm-blooded vertebrates. Thus, the presently disclosedsubject matter concerns mammals and birds.

More particularly, provided herein is the treatment of mammals such ashumans, as well as those mammals of importance due to being endangered(such as Siberian tigers), of economic importance (animals raised onfarms for consumption by humans) and/or social importance (animals keptas pets or in zoos) to humans, for instance, carnivores other thanhumans (such as cats and dogs), swine (pigs, hogs, and wild boars),ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison,and camels), and horses. Also provided is the treatment of birds,including the treatment of those kinds of birds that are endangered,kept in zoos, as well as fowl, and more particularly domesticated fowl,i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, andthe like, as they are also of economic importance to humans. Thus,provided herein is the treatment of livestock, including, but notlimited to, domesticated swine (pigs and hogs), ruminants, horses,poultry, and the like.

In some embodiments, the subject to be treated in accordance with thepresently disclosed subject matter is a subject in need of oculartreatment. In some embodiments, a subject in need of ocular comprises asubject suffering from ocular inflammation, macular edema, choroidalneovascularization, or combinations thereof.

IX. Administration

Suitable methods for administration of the compositions, therapeuticformulations and/or gene therapy constructs of the presently disclosedsubject matter include but are not limited to intravenous, subcutaneous,or intraocular injection. In some embodiments the gene therapyconstructs of the presently disclosed subject matter are administeredvia sub-Tenon injection or trans-corneal injection. Alternatively, suchtherapeutic compositions can be deposited at a site in need of treatmentin any other manner appropriate for the condition to be treated or thetarget site. For example, any approach for administration suitable forprostate tissues is within the scope of the presently disclosed subjectmatter. In some embodiments, the particular mode of administering atherapeutic composition of the presently disclosed subject matterdepends on various factors, including the distribution and abundance ofcells to be treated, the vector employed, additional tissue- orcell-targeting features of the vector, and mechanisms for metabolism orremoval of the vector from its site of administration.

X. Dose

An effective dose of the compositions, therapeutic formulations and/orgene therapy constructs of the presently disclosed subject matter isadministered to a subject in need thereof. The terms “therapeuticallyeffective amount”, “therapeutically effective dose”, “effective amount”,“effective dose” and variations thereof are used interchangeably hereinand refer to an amount of a therapeutic composition or gene therapyconstruct of the presently disclosed subject matter sufficient toproduce a measurable response (e.g. decreased HULLK expression in asubject being treated). Actual dosage levels of gene therapy constructs,and in some instances the therapeutic genes expressed by the genetherapy constructs, can be varied so as to administer an amount that iseffective to achieve the desired therapeutic response for a particularsubject. By way of example and not limitation, in some embodiments thegene therapy constructs can be administered at dose ranging from 5×10⁸to 1×10¹⁰ virus genomes (vg), which would correspond to 2×10⁸ to 5×10⁹infectious units (IFU).

In some embodiments, the dosage of the compositions, therapeuticformulations and/or gene therapy constructs of the presently disclosedsubject matter can be varied to achieve a desired level of HULLKexpression and/or activity in a subject. In some embodiments, a dosageof gene therapy construct of the presently disclosed subject matter canbe optimized to treat, prevent or reverse PCa in a subject.

In some embodiments, the quantity of a therapeutic composition of thepresently disclosed subject matter administered to a subject will dependon a number of factors including but not limited to the subject's size,weight, age, the target tissue or organ, the route of administration,the condition to be treated, and the severity of the condition to betreated.

In some embodiments the selected dosage level will depend upon theactivity of the therapeutic composition, the route of administration,combination with other drugs or treatments, the severity of thecondition being treated, and the condition and prior medical history ofthe subject being treated. However, upon a review of the instantdisclosure, it is within the skill of the art to consider these factorsin optimizing an appropriate dosage, including for example startingdoses of the compound at levels lower than required to achieve thedesired therapeutic effect and to gradually increase the dosage untilthe desired effect is achieved. Moreover, upon review of the instantdisclosure one of ordinary skill in the art can tailor the dosages to anindividual subject by making appropriate adjustments or variations, aswell as evaluation of when and how to make such adjustments orvariations, as is routine to those of ordinary skill in the art. Thepotency of a therapeutic composition can vary, and therefore a“therapeutically effective” amount can vary. However, using the assaymethods described herein below, one skilled in the art can readilyassess the potency and efficacy of a gene therapy construct of presentlydisclosed subject matter and adjust the therapeutic regimen accordingly.

EXAMPLES

The following examples are included to further illustrate variousembodiments of the presently disclosed subject matter. However, those ofordinary skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the presently disclosed subjectmatter.

Materials and Methods for Examples 1-7

Cell Culture and Reagents. LNCaP and C4-2 cells (a gift from Dr. L. W.K. Chung) were grown in DMEM:F12 (Invitrogen) with 5%Non-Heat-Inactivated serum (Gemini) and 1%Insulin-Transferrin-Selenium-Ethanolamine (ThermoFisher). CWR22Rv1(Rv1), VCaP, PC3 (gifts from Drs. Steven Balk, Karen Knudsen, and Chung,respectively), WMPY-1 (ATCC), MCF7, BT549 and MDA-MB-231 (gifts from Dr.Amy Bouton) were grown in DMEM (Invitrogen) with 10% Heat-Inactivatedserum. DU145 (a gift from Dr. Chung), PANC1 (a gift from Dr. J. ThomasParsons), HeLa and Jurkat cells (gifts from Dr. Tim Bender) were grownin RPMI-1640 (Invitrogen) with 10% Heat-Inactivated serum. LHS cells (agift from Dr. William Hahn) were grown in ProstaLife Epithelial CellMedium (Lifeline). RWPE-1 (ATCC) cells were grown inKeratinocyte-Serum-Free Media (Invitrogen). For growth and RNAexperiments, phenol-red free DMEM:F12 media with 5% Charcoal-StrippedSerum (CSS) (Gemini) was used.

Antibodies: ERK1/2 (137F5), Histone H3, LCK (73A5) (c-term), p53 (DO-7),Ran, α-Tubulin (Cell Signaling); LCK (n-term) (BD Biosciences); AR(in-house to first 21aa). Western blotting performed as previouslydescribed (17,18).

HULLK primers were based on the human LCK sequence obtained from Genbank(BC013200.1) (FIG. 1A). PCR was performed using iProof High-Fidelity DNAPolymerase (Bio-Rad) and LNCaP cDNA as the template. HULLK was ligatedinto the lentiviral expression vector PLX301 (a gift from David Root,Addgene plasmid #25895) using Gateway Cloning (ThermoFisher),transformed into DH5a competent bacteria (Life Technologies), and cloneswere sequenced for verification.

CyQuant Growth Assays: Assay was performed as previously described (18).Briefly, shRNA or pLKO control virus was added to 1 μg/mlfibronectin-coated 96 well plates. Cells were plated in RPMI-1640 plus5% CSS with vehicle, 0-0.05 nM R1881, and/or 10 μM Enzalutamide (SelleckChemicals). Quantification was performed on Day 7 using a BioTek Synergy2 plate reader.

Rapid Amplification of cDNA Ends (RACE): 5′ and 3′ RACE was performedaccording to the manufacturer's protocol (Invitrogen) using ananti-sense primer specific for LCK exon 11 (5′ RACE) and a sense primerfor LCK exon 9 (3′ RACE). 5′/3′ RACE PCR products were ligated into thepGEM sequencing vector, and multiple clones were sequenced and alignedusing the UCSC genome browser.

RNA Isolation, qPCR and ddPCR: RNA was collected using TRIzol andquantitated using a NanoDrop 2000 UV-Vis Spectrophotometer(ThermoFisher). cDNA was synthesized using Superscript IV VILO cDNAsynthesis kit (Invitrogen). Quantitative real-time PCR (qPCR) wasperformed as previously described (17,18). Droplet digital PCR (ddPCR)was executed according to the manufacturer's protocol (Bio-Rad).

Patient Samples: Formalin-fixed paraffin-embedded (FFPE) tissue wasminced and deparaffinized prior to RNA isolation. Fresh-frozen tissuewas homogenized with a mortar and pestle prior to RNA collection.

TCGA prostate cancer cohort: The LCK exon-specific RSEM data for allsamples in TCGA-PRAD cohort (32) was obtained from TSVdb (33) and thesum of RSEM values in exons 9-12 was compared to exons 1-3 to determinea 3′/5′ ratio and define HULLK expression as an increase in the 375′ratio. Clinical data for the TCGA-PRAD cohort was down loaded fromcBioPortal and LCK 3′/5′ ratio was examined comparing normal to tumorand, in tumors only, low Gleason [scores 6 and 7(3+4)] to high Gleasonscore [scores 7(4+3), and 8-10].

Example 1 Discovery of a Novel lncRNA in PCa Cells

A kinome screen was performed in LNCaP cells grown in the presence orabsence of androgen with a panel of shRNAs that targeted the kinome inorder to discover potential regulators of growth. The screen identifiedLCK as a potential positive regulator of growth. Since LCK expression isestablished in the bone marrow and immune system, but not prostate,initially testing was done for LCK expression in the hormone-sensitiveLNCaP and castration-resistant C4-2 cells by western blotting. LCKprotein expression was non-detectable in LNCaP and C4-2 cells withstandard western blots (FIG. 1A). To improve sensitivity to detect LCKprotein, immunoprecipitation using an antibody that recognized thecarboxy-terminal of the protein (LCK^(c-term)) was performed (FIG. 1A).As expected, immunoblots showed that two independent LCK antibodies,LCK^(c-term) and an amino-terminal LCK antibody (LCK^(n-term)), detectedthe 56 kDa protein in the Jurkat control cells, which expressfull-length LCK (FL-LCK). Surprisingly, LCK protein was not detected inLNCaP or C4-2 cells under FBS, CSS, or 1 nM R1881 conditions, suggestingthat LCK protein may not be expressed in these cells.

While it has been reported that LCK protein in T-cells has anapproximate 20-30 hr half-life (19), several Src family members aredownregulated following activation as a result of ubiquitination anddegradation by the proteasome (20-22). To determine whether the lack ofLCK expression was due to proteasomal degradation, LCK protein levelswere examined in the presence of the proteasome inhibitor MG132 (FIG.1B). The proteasome was successfully blocked since p53, a short-livedprotein with a 5-20 min half-life, is elevated in MG132-treated cells(23). As expected, both LCK^(c-term) and LCK^(n-term) antibodiesrecognized FL-LCK in Jurkat cells. However, LCK was still not observedat any timepoint examined in LNCaP cells treated with FBS, CSS, or 1 nMR1881. Furthermore, no truncated forms of LCK were detected when thewhole western blot membrane was probed from 75 kD to 10 kD with eitherc- or n-terminal LCK antibodies. Thus, these data suggest that LCKprotein is not expressed in PCa cells. Therefore, a ncRNA may play arole in the growth effects measured in the kinome screen.

ncRNAs represent over 70% of the human genome and are broadly dividedinto two main categories: short (<200 bases) and long (>200 bases)noncoding transcripts (16). To determine the full-length sequence of thedisclosed ncRNA, 5′ and 3′ RACE was performed (FIG. 1C). Numerous clones(n=31) were selected, purified, and sequenced. The sequence alignment ofthese clones from the 5′/3′ RACE showed that the ncRNA transcriptcompletely overlapped exon 6 through the 3′ untranslated region (3′UTR)of the LCK gene (FIG. 1D). Thus, these data show the identification of anovel lncRNA situated within the LCK gene locus that measures 1604 basesin length.

Example 2 HULLK is an AR-Regulated lncRNA

To assess whether this lncRNA is modulated by hormone, transcript levelsof this lncRNA were quantified by qPCR using 3′UTR primers in LNCaP andC4-2 cells transduced in the presence of increasing concentrations ofR1881 (FIG. 2A). A dose-dependent increase in lncRNA transcripts wasfound in response to R1881. This increase was inhibited in the presenceof two independent LCK shRNAs in both cell lines. This data suggeststhat the expression of this lncRNA is regulated by androgen.

This hormone regulation was confirmed by measuring transcript amounts inLNCaP and C4-2 cells treated with vehicle or R1881 in the presence orabsence of the anti-androgen enzalutamide (FIG. 2B). As expected, therewas an 8-fold and 12-fold R1881-induced increase in LCK 3′UTR transcriptlevels in LNCaP and C4-2 cells, respectively. Enzalutamide blocked thehormone-mediated increase in both cell lines. Androgen deprivationinfluenced message levels negligibly in LNCaP cells, but decreasedtranscript amounts of this lncRNA by approximately 2.9-fold in C4-2cells (FIG. 2B, C4-2 vehicle vs. media). There was no R1881-inducedincrease in this lncRNA in AR-null DU145 and PC3 cells, validating thatthe AR regulates the expression of this lncRNA (FIG. 2C). As a result ofthis regulation, it was decided to name this lncRNA HULLK, short forHormone Upregulated lncRNA within LCK.

AR coregulators have been shown to be influential in the progression ofprostate cancer to castration resistance (24). As disclosed herein,binding sites for two AR coactivators (p300 and Brd4) near the HULLK TSSwere discovered. p300 is known to transactivate the AR in anandrogen-dependent (25) and androgen-independent manner (26). To assesswhether p300 is involved in the regulation of HULLK expression by AR,LNCaP, C4-2, and Jurkat cells were treated with the p300 inhibitor A-485in the presence and absence of 1 nM R1881 and measured HULLK expressionby qPCR using two independent LCK primer pairs (FIG. 2D). The resultsshowed that A-485 alone had negligible effects on HULLK expression.However, the p300 inhibitor significantly opposed the hormone-inducedincrease in HULLK transcript levels. Analysis of PSA and TMPRSS2 messagein the presence of hormone and p300 inhibitor suggests that A-485 wassufficiently blocking its target. Similar effects were observed on HULLKexpression when Brd4 was suppressed with the BET family small moleculeinhibitor JQ1 (FIG. 2E). Brd4 physically associates with theamino-terminal domain of AR, and recruits RNA polymerase II to promotethe transcription of target genes (27). HULLK expression wassignificantly decreased in the presence of hormone and JQ1, compared tohormone alone. Therefore, these data together indicate that the AR mayregulate HULLK expression through the recruitment of p300 and Brd4.

Conversely, studies were conducted to determine whether HULLK modulatesAR expression. LNCaP and C4-2 cells were transduced with vector, HULLK,or shRNAs targeting LCK (n-term) (FL-LCK) or LCK (c-term) (HULLK), andAR protein and message levels were examined 48 hrs after transduction.Knockdown of HULLK did not significantly affect levels of AR protein ormRNA (FIG. 6A). Furthermore, AR expression was not notably altered bythe overexpression of HULLK (FIG. 6B). Therefore, these data suggestthat HULLK is an AR-regulated lncRNA that does not reciprocallyinfluence expression of AR itself.

Example 3 Characterization of HULLK

Since lncRNAs can be derived from many different genomic locations andtranscribed from either DNA strand, strand-specific qPCR was used todetermine which DNA strand HULLK is transcribed from. Total RNA wascollected from LNCaP cells grown in complete media, and cDNA templatewas synthesized using Oligo(dT)16, antisense strand-specific, or sensestrand-specific primers. See Table 2 for PCR primer sequences, includingHULLK cloning primers, 5′/3′ RACE primers, strand-specific PCR primers,and lncRNA localization primers. qPCR was performed with LCK Exon 11 andLCK 3′UTR primer pairs (Table 2). Amplification of PCR products fromboth LCK primer pairs was only observed with the Oligo(dT)16 and sensestrand-specific cDNA templates (FIG. 3A). However, there was nosignificant PCR amplification from either LCK primer pair when antisensestrand-specific cDNA was used as the template. These data indicate thatHULLK is transcribed from the sense strand of DNA.

TABLE 2 PCR Primer Sequences SEQ ID Sequence NO HULLK Cloning PrimersHULLK-attB1- 5′-GGGGACAAGTTTGT 2 Forward ACAAAAAAGCAGGCTTCCTGGTTCTTCAAGAACC TGAG-3′ HULLK-attB1- 5′-GGGGACAAGTTTGTA 3Start-Forward CAAAAAAGCAGGCTTCGA AGGAGATAGAACCATGGC TGGTTCTTCAAGAACCTGAG-3′ HULLK-attB2- 5′-GGGGACCACTTTGTA 4 Reverse CAAGAAAGCTGGGTCTCATCAACAGACATTTATTGA ACTC-3′ HULLK-attB2-Stop- 5′-GGGGACCACTTTGTA 5Reverse CAAGAAAGCTGGGTCCTA TCATCAACAGACATTTAT TGAACTC-3′ 5′ RACE PrimersLCK-GSP1 -Reverse 5′-CTTCGTGTGCCCGTT 6 GTAGTA-3′ LCK-GSP2-Reverse5′-CCCGAAGGTCACGAT 7 GAATAT-3′ 3′ RACE Primers LCK-GSP1-Forward5′-TACCAACTCATG 8 AGGCTGTGC-3′ LCK Sense-Strand 5′-CAGACATTTATTGA 9Primer ACTCCTGA-3′ LCK Anti-Sense- 5′-ATCGTTTTCACTG 10 Strand PrimerTCGGT-3′ LCK Primers: LCK Exon 2 (F)5-GTGTGAGAACTG 11 CCATTATC-3′(R)5′-AGAGCCATTTC 12 GGATGAG-3′ LCK Exon 4 (F)5-CAACCTGGTTAT 13CGCTCT-3′ (R)5′-CCTTCTCAAAG 14 CCCAGAT-3′ LCK Exon 11 (F)5-ATGGCATTCATT15 GAAGAGC-3′ (R)5-GTCAGACACCAG 16 AATGTTG-3′ LCK Exon 13(F)5-GGAGCTGTACCA 17 ACTCAT-3′ (R)5-CAGGTAGTCAAA 18 GGTGGG-3′ LCK 3′UTR(F)5-ATCCAGAAGTTC 19 CTCAAG-3′ (R)5′-TTACAACAGTCATC 20 AACAG-3′IncRNA Primers: DANCR (F)5′-CGGAGGTGGATTC 21 TGTTA-3′ (R)5-GTGTAGCAAGTCT22 GGTGA-3′ NEAT (F)5-GGTCTGAGGAGTG 23 ATGTG-3′ (R)5′-AAGCGTTGGTCAA 24TGTTG-3′

Some lncRNAs show preferential accumulation in the cytoplasm or nucleus,while other lncRNAs are equally expressed in both compartments. Todetermine the intracellular localization of HULLK, total RNA wascollected to synthesize cDNA from cytoplasmic and nuclear fractions ofLNCaP cells starved of hormone or exposed to 1 nM R1881 for 24 hrs andcarried out qPCR with LCK E11 and LCK 3′UTR primers to amplify HULLK.Amplification of a predominantly cytoplasmic lncRNA DANCR (28) andnuclear lncRNA NEAT1 (29) showed that transcript levels under bothconditions were substantially higher in the cytoplasm for DANCR andnucleus for NEAT1, indicating efficient cellular fractionation (FIG.3B). It was observed that hormone dramatically increased LCK E11 and LCK3′UTR transcript levels approximately 5.3-fold and 7.6-fold,respectively, in the cytoplasmic fraction, whereas the nucleartranscript amounts were approximately equivalent to the cytoplasmic CSScondition and not influenced by androgens. These results indicate thatHULLK is localized to the cytoplasm.

Example 4 HULLK Expression in PCa

Since HULLK was previously unannotated, little is known about itsexpression pattern. As the data herein shows, HULLK was discovered inPCa cells, and thus, a panel of PCa and normal prostate epithelial celllines cultured in their corresponding growth media were surveyed forHULLK expression by qPCR (FIG. 4A). Since the sequence of HULLKoverlapped LCK exon 6 through the 3′UTR, the fact that HULLK lackedexons 1-5 was exploited to distinguish HULLK expression from FL-LCK.Therefore, two LCK primer pairs—LCK 3′UTR and LCK exon 2 (LCK E2) wereutilized. LCK 3′UTR primers should amplify HULLK as well as FL-LCK;however, LCK E2 primers should only detect FL-LCK. Analysis of seven PCacell lines (LNCaP, C4-2, CWR22Rv1, PC3, DU145, VCaP, LAPC4) and threenormal prostate cell lines (WMPY1, RWPE, LHS) revealed that HULLK isexpressed to varying degrees in all cell lines examined, as PCRamplification was only detected with the LCK 3′UTR primers and not theLCK E2. As a control for primer efficiency, FL-LCK was detected inJurkat cells with both primer pairs (data not shown). These data showthat HULLK can be successfully detected by employing this qPCR method oftwo primer pairs targeting two specific regions in LCK—exons 1-5 andexon 6-3′UTR.

HULLK expression was also examined in other cancer tissue types and notranscripts were observed in cervical (HeLa) or pancreatic (PANC1)cancer cell lines grown under normal serum conditions (FIG. 4B). HULLKwas detected in the ER+AR+ luminal A subtype MCF7 breast cancer (BCa)cell line but not in the triple negative Claudin-low BT549 or MDA-MB-231BCa cell lines (31).

Applying the two LCK primer pair method, HULLK levels were determined intwenty-six FFPE PCa tissue samples from patients presenting with Gleasonscore 6-10 disease and seven normal prostate tissue obtained from theUniversity of Virginia Biorepository and Tissue Research Facility (FIG.4C). The results were displayed as the ratio of SQ mean of thecarboxy-terminal primer pairs to the amino-terminal primer pair, where<1 indicates FL-LCK and >1 indicates more HULLK. HULLK expression wasnot detected in the normal prostate tissue that were examined. However,a significant positive correlation was found between HULLK expressionand higher Gleason score. This correlation was confirmed using a secondcohort of sixteen fresh-frozen PCa tissue from the University of TexasSouthwestern (FIG. 4D). In this cohort, similar results were discoveredas the first cohort; there was a significant increase in HULLKexpression with increased Gleason score. Statistical analyses revealedthat pooling the data from the two cohorts does not diminish thesignificance of this correlation between HULLK levels and Gleason score(FIG. 4E). Finally, the PRAD TCGA cohort (32) was interrogated for HULLKexpression and association with clinical correlate. The LCKexon-specific RSEM data for all samples in this cohort was obtained fromTSVdb (33), to calculate the 3′/5′ ratio and define HULLK expression asan increase in the 3′/5′ ratio. An increase in HULLK expression wasfound when comparing normal to tumor and, in tumors only, low Gleason[scores 6 and 7(3+4)] to high Gleason score [scores 7(4+3), and 8-10].Interestingly, very high 3′/5′ ratios were found in the PRAD TCGAdataset. However, applying a multivariate Cox Proportional Hazardsmodel, controlling for age and Gleason grade, the data did not revealwhether HULLK expression alone was significantly associated with shortertime to biochemical recurrence (data not shown). These data stronglyindicate that HULLK is expressed in PCa patients and upregulated withadvanced disease.

Example 5 HULLK Positively Regulates PCa Cell Growth

To explore the functional role of HULLK in PCa, the effects of HULLKknockdown on PCa cell growth was examined. LNCaP, C4-2, and Rv1 cellswere transduced with lentiviral particles expressing four independentshRNAs specific for LCK or pLKO empty vector control in the presence orabsence of 0.05 nM R1881. Two shRNAs were targeting the carboxy-terminalof LCK (shLCK-3 and shLCK-4) and should decrease HULLK levels; and twoshRNAs were directed toward the amino-terminal (shLCK-1 and shLCK-2) andshould not affect HULLK expression (FIG. 5A). It was confirmed by qPCRthat shLCK-3 and shLCK-4 decreased HULLK expression by 70-90% in LNCaP,C4-2, and Rv1 cells (FIG. 5B). shLCK-1 and shLCK-2 had little to noeffect on HULLK expression in these three cell lines. As a control, allfour shRNAs efficiently knocked LCK down 60-90% in Jurkat cells. Sevendays following viral transduction, cellular growth was measured usingCyQuant, which uses DNA content as a surrogate for cell number. In theabsence of hormone, the four shRNAs had no dramatic effects on growth inLNCaP or C4-2 cells (FIG. 5C). However, there was a statisticallysignificant decrease in cell growth in Rv1 cells with shLCK-2, shLCK-3,and shLCK-4. In the presence of 0.05 nM R1881, the carboxy-terminalshRNAs significantly inhibited growth of LNCaP, C4-2, and Rv1 cellscompared to the pLKO control cells. While the amino-terminal shRNAs didnot have any significant effects on growth in LNCaP or C4-2 cells, therewas a notable proliferative decrease in Rv1 cells. These data suggestthat HULLK may drive PCa cell growth.

The complement experiment of evaluating the effects of overexpressingHULLK on cell growth was also performed. LNCaP, C4-2, and Rv1 cells weretransduced with lentivirus expressing either vector or HULLK constructsin the presence or absence of 0.05 nM R1881, and proliferation wascalculated seven days following viral transduction. Overexpression ofHULLK did not provide a growth advantage over vector-expressing cells inandrogen-deprived media (data not shown). The expected increase in cellgrowth in all vector-expressing control cells exposed to R1881 (FIG. 5D)was observed. However, hormone significantly increased proliferation inHULLK-overexpressing cells compared to vector control cells, suggestingthat there may be a greater sensitivity to hormone when HULLK isoverexpressed in PCa cells. Together, these data indicate that HULLK isa positive regulator of PCa cell growth and may help drive CRPC byincreasing sensitivity to hormone.

Example 6 Discussion of Experimental Results

One in forty-one men diagnosed with PCa will die from the disease. WhileADT is the current initial treatment for advanced PCa, eventually allmen diagnosed with PCa will develop incurable CRPC. Therefore, thereexists a serious need for more effective therapies for the treatment ofadvanced PCa, and that requires a more complete understanding of PCadevelopment and progression. Described herein is a previouslyunannotated lncRNA, referred to as HULLK for Hormone Upregulated lncRNAwithin LCK, that functions as a positive regulator of PCa cells and theexpression of which correlates with high grade PCa.

Genome sequencing has led to the surprising discovery thatprotein-coding RNAs only make up approximately 2% of the human genome,while ncRNAs represents 70-90% (34). Once believed to be transcriptionalnoise, ncRNAs are now associated with many normal biological processes,including transcriptional and translational regulation, chromatinmodification, and cell cycle regulation. Furthermore, accumulatingevidence supports vital roles in cancer initiation, development, andprogression for ncRNAs (35,36), which are generically divided into twogroups: small and long noncoding transcripts. While small ncRNAs,especially microRNAs, have been well-documented to play important rolesin human disease by regulating the expression of target mRNA, lncRNAsare only beginning to be investigated and scrutinized. Even thoughthousands of lncRNAs have been annotated in the human genome, fewfunctional lncRNAs in PCa have been fully characterized.

Disclosed herein is a novel lncRNA in PCa from a high-throughput RNAiscreen to uncover potential regulators of PCa cell growth. LCK wasidentified from the shRNA screen as a positive regulator of PCa cellularproliferation. However, LCK protein expression was never detected.Immunoprecipitation and Western analyses revealed that LCK could only beobserved in control Jurkat cells and not LNCaP or C4-2 PCa cell lines.The inability to recognize LCK in PCa cells was not due to the short LCKhalf-life, as the proteasome inhibitor MG132 did not facilitate thedetection of LCK protein. The open reading frame (ORF) finder from theNational Center for Biotechnology Information predicted that there wereone long and two short ORFs in the HULLK sequence. According to theGenScript Codon Bias tool, the two short ORFs have low codon adaptationindices (CAI) (<0.8), suggesting poor expression. The long ORF, which isin frame with LCK and corresponds to the kinase domain, has a CAI (0.83)similar to LCK exons 1-5, suggesting that this ORF calculated to producea 28.59 kDa protein has an equal chance of being expressed as exons 1-5of LCK. Furthermore, data from the FANTOM Project suggested that therewas a TSS near LCK exon 12, resulting in an estimated 13 kDa truncatedLCK protein (39). Using an antibody raised against tyrosine 505 in exon13 (kinase domain) of human LCK, the predicted 28.59 kDa or 13 kDa LCKprotein or any other shorter LCK isoforms were able to be detected. Thisantibody did recognize LCK in Jurkat cells. These data suggested thatLCK protein was not expressed in PCa cells. The disclosed data areconsistent with the ChIP-seq dataset from the ENCODE Project, where theH3K4me3 peaks were highly enriched at active promoters near TSSs inJurkat cells (40). Therefore, the shRNAs utilized in the kinome screenmay have targeted a noncoding RNA within the LCK gene locus.

5′/3′ RACE was utilized to uncover a novel noncoding transcript thataligned with the LCK gene and completely overlapped exons 6-13 and 3′UTRon the sense strand of DNA. All of the clones examined from the 3′ RACEshowed the same end, but several clones from the 5′ RACE varied in theends by roughly 5-20 nucleotides. The presence of a 5′-3′exoribonuclease in the PCR reaction likely account for these differencesin 5′ ends.

Ning and colleagues reanalyzed the dataset from the Necsulea et al study(41) and reported that 29.3% of 24,793 annotated lncRNAs overlappedprotein-coding genes (42). The overlapping lncRNAs were categorized intofive main groups based on DNA strand: overlapping on opposite strands(5′-regions overlap, 3′-regions overlap, embedded pairs) and overlappingon same strand (5′-regions overlap with 3′-regions, embedded pairs). Theembedded pairs on either strand represented 76.4% of all annotatedoverlapping lncRNA-protein-coding pairs. The majority of the overlaps(˜93%) occurred on the opposite DNA strand. However, lncRNAs embedded ina protein-coding gene on the same DNA strand only amounted to 5.6%.Similar to the fact that overlapping genes tend to be coexpressed (43),lncRNA-protein-coding pairs exhibited an overall positive expressioncorrelation, with the Spearman coefficient for same strand overlapsbeing greater than opposite strand overlaps (42). These observationssuggested that the HULLK-LCK gene pair may be rare, since theyoverlapped on the same strand and the protein-coding partner is notexpressed in PCa cells.

The cellular compartment in which a particular lncRNA is localized maybe used as a cue to its functions. Nuclear lncRNAs can facilitateepigenetic regulation through the binding of chromatin and chromatinmodifying proteins (44), whereas cytoplasmic lncRNAs can influenceseveral cellular processes, including protein degradation andtranscription (45). Much less is known about cytoplasmic lncRNAscompared to their nuclear counterparts. The disclosed work on HULLKaugments the growing list of cytoplasmic lncRNA functions. Thefunctional role of HULLK in PCa was explored by knocking down HULLK withtwo independent shRNAs in LNCaP, C4-2, and Rv1 cells. There was asignificant decrease in cell growth in the absence and presence ofandrogens when HULLK was depleted from the cell. HULLK overexpressionresulted in a modest but consistent increase in cellular proliferationin response to hormone. These results suggested that HULLK is oncogenicin nature, similar to other lncRNAs in PCa, including PCAT-1 (45), PCA3(3), and SChLAP1 (46).

The expression of lncRNAs typically has been cell-type and tissuespecific (1). Furthermore, lncRNAs that overlap protein-coding genesshowed higher tissue specificity than non-overlappinglncRNA-protein-coding gene pairs (42). The data disclosed herein weremainly consistent with the tissue specificity of lncRNAs, includingobservation of HULLK expression in a panel of PCa cell lines and tissue.HULLK transcripts were not quantitated above transcriptional noise incervical or pancreatic cancer cell lines. However, HULLK transcriptswere discovered in MCF7 cells and not BT549 or MDA-MB-231 cells. Whileall three of the BCa lines are reported to express AR (47,48), in theselaboratory tests only AR protein levels are only detected in MCF7 andnot BT549 or MDB-MB-231 cells (FIG. 7A). Furthermore, HULLK expressionincreases in response to 1 nM R1881 in MCF7 cells, but not BT549 orMDA-MB-231 cells (FIG. 7B). In addition to the prostate specificity ofHULLK, these data also suggest that HULLK may be expressed in othertissues where AR is active. Akin to PCGEM1 (49), CTBP1-AS (11), and PCA3(3) lncRNAs, HULLK was upregulated in response to androgen. Moreimportantly, expression of HULLK was significantly upregulated inhigh-grade PCa specimens in three cohorts, including the PCa TCGA.

Since LCK-expressing lymphocytes infiltrate prostate tumors (50), thefact that HULLK lacked LCK exons 1-5 was exploited, and the ratio ofc-terminal LCK primers:n-terminal primers (HULLK:FL-LCK) was used todistinguish HULLK expression from LCK. It was observed that the ratiosfor the FFPE samples were higher than the fresh frozen tissue. Theseratios could be influenced by differences in tissue collection andcomposition. For the FFPEs, the extent of infiltrating lymphocytecontamination was reduced by demarcating portions of tumor fromsurrounding lymphocyte-containing stroma. Decreasing the stromalcontribution would diminish the amount of LCK, and thus, increase theratios of HULLK:LCK. Even with the compounded presence of lymphocytes inthe fresh frozen cohort, HULLK expression still displayed a significantupregulation with increasing Gleason score. Examining the TCGA data forthe 3′/5′ ratio of LCK validated this observation, which showedincreasing HULLK expression in tumor compared to normal, and withintumors, with higher Gleason score compared to low Gleason score. Thesame finding of HULLK expression correlating with Gleason score in threeindependent cohorts, when considered with the experimental data thatHULLK over expression increases growth and HULLK knockdown decreases PCagrowth in androgen-dependent and castration-resistant PCa lines,strongly suggests that HULLK expression is associated with aggressivedisease and may be a driver of PCa.

As disclosed herein, a potential mechanism for growth stimulation byHULLK was explored. Since HULLK is embedded in the LCK gene locus, itwas first hypothesized that HULLK may be regulating cell growth throughthe modulation of the expression of Src family kinases. Knockdown ofHULLK with shRNAs revealed that the levels of protein and message ofeach Src family member examined (Blk, Fgr, Frk, Fyn, Hck, Lyn, Src, andYes) were not significantly altered, suggesting that HULLK may notaffect cell proliferation through the regulation of Src family kinaseexpression (data not shown).

lncRNAs can exert their effects locally by regulating the expression ofneighboring genes in cis or distantly in trans (51). There are two genesimmediately 5′ (Eukaryotic translation initiation factor 3 subunit I,EIF3I) and 3′ (histone deacetylase 1, HDAC1) of HULLK with reportedbiological functions that may influence cell growth. EIF3I plays acrucial role in the initiation of protein synthesis, whereas HDAC1interacts with the retinoblastoma tumor-suppressor protein to controlcell proliferation and differentiation (52). To determine whether theeffects on cell growth mediated by HULLK were a result of the regulationof gene expression in cis, the transcript levels of EIF3I and HDAC1 weremeasured in HULLK-depleted PCa cells. It was found that HULLK knockdowndid not dramatically change the amounts of message of either gene,suggesting that the regulation of gene expression in cis, at least forthese two genes, may not account for the HULLK-mediated effects on cellgrowth (data not shown). While no significant alterations in these genesat the RNA level was observed, the possibility that HULLK may beaffecting these genes at the protein level cannot be ruled out. lncRNAscan function as scaffolds or sponges, thereby influencing translationefficiency, cellular localization, or protein stability (51).

Example 7 Conclusions

Disclosed herein is a novel lncRNA completely overlapping exons 6-13 and3′UTR of the LCK gene on 1p35.1. The data disclosed herein establishthat this lncRNA is regulated by androgens and the AR, and as such, thislncRNA has been named “HULLK” for Hormone Upregulated lncRNA within LCK.The instant disclosure reveals that HULLK is localized to the cytoplasmand found on the sense strand within the LCK gene. HULLK is expressed inPCa cell lines and upregulated as PCa progresses to metastatic disease.As demonstrated herein, HULLK functions as an oncogene to positivelyregulate PCa cell proliferation. Thus, the data herein enhances theunderstanding of lncRNA biology and may assist in the development ofadditional biomarkers or more effective therapeutic targets for advancedPCa.

REFERENCES

All references listed herein including but not limited to all patents,patent applications and publications thereof, scientific journalarticles, and database entries (e.g., GENBANK® database entries and allannotations available therein) are incorporated herein by reference intheir entireties to the extent that they supplement, explain, provide abackground for, or teach methodology, techniques, and/or compositionsemployed herein.

-   1. Prensner J R, Chinnaiyan A M. The emergence of lncRNAs in cancer    biology. Cancer Discov [Internet]. 2011 October; 1(5):391-407.-   2. Xu S, Yi X-M, Tang C-P, Ge J-P, Zhang Z-Y, Zhou W-Q. Long    non-coding RNA ATB promotes growth and epithelial-mesenchymal    transition and predicts poor prognosis in human prostate carcinoma.    Oncol Rep [Internet]. 2016 May 9 [cited 2017 Jul. 3]; 36(1):10-22.-   3. Lemos AEG, Ferreira L B, Batoreu N M, de Freitas P P, Bonamino M    H, Gimba ERP. PCA3 long noncoding RNA modulates the expression of    key cancer-related genes in LNCaP prostate cancer cells. Tumor Biol    [Internet]. 2016 Aug. 9 [cited 2017 Jul. 3]; 37(8):11339-48.-   4. Salameh A, Lee A K, Card?-Vila M, Nunes D N, Efstathiou E,    Staquicini F I, et al. PRUNE2 is a human prostate cancer suppressor    regulated by the intronic long noncoding RNA PCA3. Proc Natl Acad    Sci [Internet]. 2015 Jul. 7 [cited 2017 Jul. 5]; 112(27):8403-8.-   5. Kretz M, Webster D E, Flockhart R J, Lee C S, Zehnder A,    Lopez-Pajares V, et al. Suppression of progenitor differentiation    requires the long noncoding RNA ANCR. Genes Dev [Internet]. 2012    Feb. 15 [cited 2017 Jul. 5]; 26(4):338-43.-   6. Heer R, Robson C N, Shenton B K, Leung H Y. The role of androgen    in determining differentiation and regulation of androgen receptor    expression in the human prostatic epithelium transient amplifying    population. J Cell Physiol [Internet]. 2007 September [cited 2017    Jul. 5]; 212(3):572-8.-   7. Ma W-L, Jeng L-B, Lai H-C, Liao P-Y, Chang C. Androgen receptor    enhances cell adhesion and decreases cell migration via modulating    β1-integrin-AKT signaling in hepatocellular carcinoma cells. Cancer    Lett [Internet]. 2014 Aug. 28 [cited 2017 Jul. 5]; 351(1):64-71.-   8. Jia J, Li F, Tang X-S, Xu S, Gao Y, Shi Q, et al. Long noncoding    RNA DANCR promotes invasion of prostate cancer through    epigenetically silencing expression of TIMP2/3. Oncotarget    [Internet]. 2014 Nov. 9 [cited 2017 Jul. 5]; 7(25):37868-81.-   9. Malik R, Patel L, Prensner J R, Shi Y, Iyer M K, Subramaniyan S,    et al. The lncRNA PCAT29 Inhibits Oncogenic Phenotypes in Prostate    Cancer. Mol Cancer Res [Internet]. 2014 August [cited 2014 Aug. 28];    12(8):1081-7.-   10. Zhang Z, Zhou N, Huang J, Ho T-T, Zhu Z, Qiu Z, et al.    Regulation of androgen receptor splice variant AR3 by PCGEM1.    Oncotarget [Internet]. 2016 Mar. 29 [cited 2017 Jul. 5];    7(13):15481-91.-   11. Takayama K, Horie-Inoue K, Katayama S, Suzuki T, Tsutsumi S,    Ikeda K, et al. Androgen-responsive long noncoding RNA CTBP1-A S    promotes prostate cancer. EMBO J [Internet]. 2013 May 3 [cited 2018    Apr. 7]; 32(12):1665-80.-   12. Zhang A, Zhao J C, Kim J, Fong K, Yang Y A, Chakravarti D, et    al. LncRNA HOTAIR Enhances the Androgen-Receptor-Mediated    Transcriptional Program and Drives Castration-Resistant Prostate    Cancer. Cell Rep [Internet]. 2015 Oct. 6 [cited 2017 Jul. 5];    13(1):209-21.-   13. Chakravarty D, Sboner A, Nair S S, Giannopoulou E, Li R, Hennig    S, et al. The oestrogen receptor alpha-regulated lncRNA NEAT1 is a    critical modulator of prostate cancer. Nat Commun [Internet]. 2014    Nov. 21 [cited 2018 Apr. 5]; 5:5383.-   14. Ylipää A, Kivinummi K, Kohvakka A, Annala M, Latonen L,    Scaravilli M, et al. Transcriptome Sequencing Reveals PCAT5 as a    Novel ERG-Regulated Long Noncoding RNA in Prostate Cancer. Cancer    Res [Internet]. 2015 Oct. 1 [cited 2018 Nov. 16]; 75(19):4026-31.-   15. Prensner J R, Iyer M K, Sahu A, Asangani I A, Cao Q, Patel L, et    al. The long noncoding RNA SChLAP1 promotes aggressive prostate    cancer and antagonizes the SWI/SNF complex. Nat Genet [Internet].    2013 November [cited 2014 Apr. 21]; 45(11):1392-8.-   16. Derrien T, Johnson R, Bussotti G, Tanzer A, Djebali S, Tilgner    H, et al. The GENCODE v7 catalog of human long noncoding RNAs:    Analysis of their gene structure, evolution, and expression. Genome    Res [Internet]. 2012 Sep. 1 [cited 2018 Nov. 15]; 22(9):1775-89.-   17. Gordon V, Bhadel S, Wunderlich W, Zhang J, Ficarro S B, Mollah S    A, et al. CDK9 regulates A R promoter selectivity and cell growth    through serine 81 phosphorylation. Mol Endocrinol [Internet]. 2010    December [cited 2012 Mar. 13]; 24(12):2267-80.-   18. Whitworth H, Bhadel S, Ivey M, Conaway M, Spencer A, Hernan R,    et al. Identification of Kinases Regulating Prostate Cancer Cell    Growth Using an RNAi Phenotypic Screen. PLoS One [Internet]. 2012    June [cited 2013 Sep. 11]; 7(6).-   19. Hurley P J, Bunz F. ATM and ATR: components of an integrated    circuit. Cell Cycle [Internet]. 2007 February; 6(4):414-7.-   20. Harris K F, Shoji I, Cooper E M, Kumar S, Oda H, Howley P M.    Ubiquitin-mediated degradation of active Src tyrosine kinase. Proc    Natl Acad Sci USA [Internet]. 1999 Nov. 23 [cited 2017 Jun. 28];    96(24):13738-43.-   21. Oda H, Kumar S, Howley P M. Regulation of the Src family    tyrosine kinase Blk through E6A P-mediated ubiquitination. Proc Natl    Acad Sci USA [Internet]. 1999 Aug. 17 [cited 2017 Jun. 28];    96(17):9557-62.-   22. Rao N, Miyake S, Reddi A L, Douillard P, Ghosh A K, Dodge I L,    et al. Negative regulation of Lck by Cbl ubiquitin ligase. Proc Natl    Acad Sci USA [Internet]. 2002 Mar. 19 [cited 2017 Jun. 28];    99(6):3794-9.-   23. Giaccia A J, Kastan M B. The complexity of p53 modulation:    emerging patterns from divergent signals. Genes Dev [Internet]. 1998    Oct. 1 [cited 2017 Jun. 28]; 12(19):2973-83.-   24. Heemers H V., Tindall D J. Androgen Receptor (A R) Coregulators:    A Diversity of Functions Converging on and Regulating the A R    Transcriptional Complex. Endocr Rev [Internet]. 2007 December [cited    2014 Mar. 13]; 28(7):778-808.-   25. Fu M, Wang C, Reutens A T, Wang J, Angeletti R H, Siconolfi-Baez    L, et al. p300 and p300/cAMP-response element-binding    protein-associated factor acetylate the androgen receptor at sites    governing hormone-dependent transactivation. J Biol Chem [Internet].    2000 Jul. 7 [cited 2018 Oct. 11]; 275(27):20853-60.-   26. Debes J D, Tindall D J. The role of androgens and the androgen    receptor in prostate cancer. Cancer Lett [Internet]. 2002 Dec. 10    [cited 2018 Oct. 11]; 187(1-2):1-7. A-   27. Asangani I A, Dommeti V L, Wang X, Malik R, Cieslik M, Yang R,    et al. Therapeutic targeting of BET bromodomain proteins in    castration-resistant prostate cancer. Nature [Internet]. 2014 Jun.    23 [cited 2018 Oct. 11]; 510(7504):278-82.-   28. van Heesch S, van Iterson M, Jacobi J, Boymans S, Essers P B, de    Bruijn E, et al. Extensive localization of long noncoding RNAs to    the cytosol and mono- and polyribosomal complexes. Genome Biol    [Internet]. 2014 January [cited 2015 Jan. 17]; 15(1):R6.-   29. Clemson C M, Hutchinson J N, Sara S A, Ensminger A W, Fox A H,    Chess A, et al. An architectural role for a nuclear noncoding RNA:    NEAT1 RNA is essential for the structure of paraspeckles. Mol Cell    [Internet]. 2009 Mar. 27 [cited 2017 Jun. 30]; 33(6):717-26.-   30. Brunner A L, Beck A H, Edris B, Sweeney R T, Zhu S X, Li R, et    al. Transcriptional profiling of long non-coding RNAs and novel    transcribed regions across a diverse panel of archived human    cancers. [cited 2017 Jun. 30];-   31. Holliday D L, Speirs V. Choosing the right cell line for breast    cancer research. Breast Cancer Res [Internet]. 2011 Aug. 12 [cited    2018 Nov. 2]; 13(4):215.-   32. Abeshouse A, Ahn J, Akbani R, Ally A, Amin S, Andry C D, et al.    The Molecular Taxonomy of Primary Prostate Cancer. Cell [Internet].    2015 November [cited 2019 May 9]; 163(4):1011-25.-   33. Sun W, Duan T, Ye P, Chen K, Zhang G, Lai M, et al. TSVdb: a    web-tool for TCGA splicing variants analysis. BMC Genomics    [Internet]. 2018 May 29 [cited 2019 May 9]; 19(1):405.-   34. ENCODE Project Consortium. An integrated encyclopedia of DNA    elements in the human genome. Nature [Internet]. 2012 Sep. 6 [cited    2018 Apr. 2]; 489(7414):57-74.-   35. Alexander R P, Fang G, Rozowsky J, Snyder M, Gerstein M B.    Annotating non-coding regions of the genome. Nat Rev Genet    [Internet]. 2010 Aug. 13 [cited 2018 Apr. 1]; 11 (8): 559-71.-   36. Geisler S, Coller J. RNA in unexpected places: long non-coding    RNA functions in diverse cellular contexts. Nat Rev Mol Cell Biol    [Internet]. 2013 Nov. 9 [cited 2018 Apr. 1]; 14(11):699-712.-   37. Genomics B, Hutchinson J N, Ensminger A W, Clemson C M, Lynch C    R, Lawrence J B, et al. A screen for nuclear transcripts identifies    two linked noncoding RNAs associated with SC35 splicing domains. BMC    Genomics [Internet]. 2007 [cited 2017 Jun. 29]; 8(8).-   38. Yang L, Lin C, Jin C, Yang J C, Tanasa B, Li W, et al.    lncRNA-dependent mechanisms of androgen-receptor-regulated gene    activation programs. Nature [Internet]. 2013 August;-   39. Lizio M, Harshbarger J, Shimoji H, Severin J, Kasukawa T, Sahin    S, et al. Gateways to the FANTOMS promoter level mammalian    expression atlas. Genome Biol [Internet]. 2015 Jan. 5 [cited 2018    Apr. 2]; 16(1):22.-   40. Benayoun B A, Pollina E A, Ucar D, Mahmoudi S, Karra K, Wong E    D, et al. H3K4me3 Breadth Is Linked to Cell Identity and    Transcriptional Consistency. Cell [Internet]. 2015 Nov. 19 [cited    2018 Nov. 15]; 163(5):1281-6.-   41. Necsulea A, Soumillon M, Warnefors M, Liechti A, Daish T, Zeller    U, et al. The evolution of lncRNA repertoires and expression    patterns in tetrapods. Nature [Internet]. 2014 Jan. 19 [cited 2018    Apr. 4]; 505(7485):635-40.-   42. Ning Q, Li Y, Wang Z, Zhou S, Sun H, Yu G. The Evolution and    Expression Pattern of Human Overlapping lncRNA and Protein-coding    Gene Pairs. Sci Rep [Internet]. 2017 Dec. 27 [cited 2018 Nov. 15];    7(1):42775.-   43. Balbin O A, Malik R, Dhanasekaran S M, Prensner J R, Cao X, Wu    Y-M, et al. The landscape of antisense gene expression in human    cancers. Genome Res [Internet]. 2015 July [cited 2018 Apr. 6];    25(7):1068-79.-   44. Lee J T. Epigenetic Regulation by Long Noncoding RNAs. Science    (80-) [Internet]. 2012 Dec. 14 [cited 2018 Nov. 16];    338(6113):1435-9.-   45. Prensner J R, Chen W, Han S, Iyer M K, Cao Q, Kothari V, et al.    The long non-coding RNA PCAT-1 promotes prostate cancer cell    proliferation through cMyc. Neoplasia [Internet]. 2014 November    [cited 2018 Apr. 5]; 16(11):900-8.-   46. Prensner J R, Iyer M K, Sahu A, Asangani I A, Cao Q, Patel L, et    al. The long noncoding RNA SChLAP1 promotes aggressive prostate    cancer and antagonizes the SWI/SNF complex. Nat Genet [Internet].    2013 [cited 2017 Jun. 29]; 11.-   47. Macedo L F, Guo Z, Tilghman S L, Sabnis G J, Qiu Y, Brodie A.    Role of Androgens on MCF-7 Breast Cancer Cell Growth and on the    Inhibitory Effect of Letrozole. Cancer Res [Internet]. 2006 Aug. 1    [cited 2018 Nov. 2]; 66(15):7775-82.-   48. Barton V N, D'Amato N C, Gordon M A, Lind H T, Spoelstra N S,    Babbs B L, et al. Multiple molecular subtypes of triple-negative    breast cancer critically rely on androgen receptor and respond to    enzalutamide in vivo. Mol Cancer Ther [Internet]. 2015 March [cited    2018 Nov. 2]; 14(3):769-78.-   49. Parolia A, Crea F, Xue H, Wang Y, Mo F, Ramnarine V, et al. The    long non-coding RNA PCGEM1 is regulated by androgen receptor    activity in vivo. Mol Cancer [Internet]. 2015 Feb. 21 [cited 2018    Apr. 7]; 14(1):46.-   50. Rädestad E, Egevad L, Jorns C, Mattsson J, Sundberg B, Nava S,    et al. Characterization of infiltrating lymphocytes in human benign    and malignant prostate tissue. Oncotarget [Internet]. 2017 Sep. 1    [cited 2018 Apr. 8]; 8(36):60257-69.-   51. Akhade V S, Pal D, Kanduri C. Long Noncoding RNA: Genome    Organization and Mechanism of Action. In: Advances in experimental    medicine and biology [Internet]. 2017 [cited 2019 Apr. 24]. p.    47-74.-   52. Magnaghi-Jaulin L, Groisman R, Naguibneva I, Robin P, Lorain S,    Le Villain J P, et al. Retinoblastoma protein represses    transcription by recruiting a histone deacetylase. Nature    [Internet]. 1998 Feb. 5 [cited 2019 Apr. 24]; 391(6667):601-5.

It will be understood that various details of the presently disclosedsubject matter may be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

What is claimed is:
 1. A method of modulating lymphocyte-specificprotein tyrosine kinase (LCK) activity in a vertebrate subject, themethod comprising: administering to the vertebrate subject an effectiveamount of a substance capable of modulating expression of an LCK gene inthe vertebrate subject, wherein the substance comprises an RNAinterference (RNAi) molecule directed to the LCK gene, wherebymodulation of LCK activity is accomplished and whereby modulatingexpression of the LCK gene modulates growth and/or proliferation of aprostate cancer (PCa) cell within the vertebrate subject, wherein theRNAi molecule comprises a short hairpin RNA (shRNA), whereby the shRNAmodulates expression of the LCK gene by RNAi, and wherein the shRNAcomprises shLCK-3 and/or shLCK-4.
 2. The method of claim 1, whereinmodulating expression of the LCK gene comprises modulating expression ofa long noncoding RNA (lncRNA) of the LCK gene.
 3. The method of claim 2,wherein the lncRNA comprises a hormone upregulated lncRNA within the LCKgene (HULLK).
 4. The method of claim 3, wherein HULLK comprises anucleotide sequence having at least about 75% sequence identity to SEQID NO.
 1. 5. The method of claim 1, wherein the shRNA is configured totarget a carboxy-terminal of the LCK gene.
 6. The method of claim 1,wherein the substance further comprises an anti-androgen compound. 7.The method of claim 1, wherein the substance further comprises adelivery vehicle, wherein the delivery vehicle comprises a viral vector,an antibody, an aptamer and/or a nanoparticle, for delivering the shRNAto a target cell.
 8. The method of claim 1, wherein the RNAi moleculecomprises a small interfering RNA (siRNA), whereby the siRNA modulatesexpression the LCK gene by RNAi.
 9. The method of claim 8, wherein thesubstance further comprises a delivery vehicle, wherein the deliveryvehicle is selected from a viral vector, an antibody, an aptamer, or ananoparticle for delivering the siRNA to a target cell.
 10. The methodof claim 3, wherein the substance is configured to target HULLK incytoplasm of a cell in the vertebrate subject.
 11. A method ofmodulating lymphocyte-specific protein tyrosine kinase (LCK) activity ina vertebrate subject, the method comprising: administering to thevertebrate subject an effective amount of a substance capable ofmodulating expression of an LCK gene in the vertebrate subject andwherein the vertebrate subject is suffering from prostate cancer (PCa),wherein the PCa comprises androgen-dependent PCa and/orcastration-resistant PCa, wherein the substance comprises an RNAinterference (RNAi) molecule directed to the LCK gene, wherebymodulation of LCK activity is accomplished, and wherein the RNAimolecule comprises a short hairpin RNA (shRNA), whereby the shRNAmodulates expression of the LCK gene by RNAi, and wherein the shRNAcomprises shLCK-3 and/or shLCK-4.
 12. The method of claim 11, whereinmodulating expression of the LCK gene comprises modulating expression ofa long noncoding RNA (lncRNA) of the LCK gene.
 13. The method of claim12, wherein the lncRNA comprises a hormone upregulated lncRNA within theLCK gene (HULLK).
 14. The method of claim 13, wherein HULLK comprises anucleotide sequence having at least about 75% sequence identity to SEQID NO.
 1. 15. The method of claim 11, wherein the shRNA is configured totarget a carboxy-terminal of the LCK gene.
 16. The method of claim 11,wherein the substance further comprises an anti-androgen compound. 17.The method of claim 16, wherein the anti-androgen compound is selectedfrom the group consisting of enzalutamide, an inhibitor of p300, aninhibitor of the bromodomain family, and an inhibitor of theextra-terminal (BET) family.
 18. The method of claim 11, wherein thesubstance further comprises a delivery vehicle, wherein the deliveryvehicle comprises a viral vector, an antibody, an aptamer and/or ananoparticle, for delivering the shRNA to a target cell.
 19. The methodof claim 11, wherein the RNAi molecule comprises a small interfering RNA(siRNA), whereby the siRNA modulates expression the LCK gene by RNAi.20. The method of claim 18, wherein the substance further comprises adelivery vehicle, wherein the delivery vehicle is selected from a viralvector, an antibody, an aptamer, or a nanoparticle for delivering thesiRNA to a target cell.
 21. The method of claim 13, wherein thesubstance is configured to target HULLK in cytoplasm of a cell in thevertebrate subject.
 22. The method of claim 6, wherein the anti-androgencompound is selected from the group consisting of enzalutamide, aninhibitor of p300, an inhibitor of the bromodomain family, and aninhibitor of the extra-terminal (BET) family.